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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.

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.

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.

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 pre-clinical applications.

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.

INTRODUCTION The median survival of glioblastoma (GBM) patients remains less than two years even with state-of-the-art treatment. Current targeted GBM therapies demonstrate initial therapeutic benefit; however, patients relapse due to therapeutic selection of treatment resistant GBM cellular populations. Therefore, we propose targeting pathologic disruption of the blood brain barrier (BBB) via exposure of neural ECM, rather than disease markers, to overcome therapy-resistant GBM. METHODS We identify Variable Lymphocyte Receptors (VLRs, the antigen recognition system used by lamprey) that demonstrate high specificity for neural ECM. Candidate VLRs underwent further refinement using in vitro binding assays and ex vivo tissue staining. Utilizing pathologic disruption of BBB as an approach for targeting GBM was confirmed in vivo using intracranial murine glioblastoma models. RESULTS The lead neural ECM-binding VLR candidate, named P1C10, demonstrates diffuse binding to parenchymal neural ECM, without detectable binding to other tissues. P1C10 demonstrates nanomolar affinity for in vitro derived neural ECM, and preferentially accumulates at intracranial GL261 and U87 lesions in murine GBM models. Finally, administration of P1C10-targeted doxorubicin-loaded liposomes significantly extends the survival of mice bearing intracranial U87 GBM. CONCLUSIONS We identified VLRs that bind neural ECM, and demonstrate their utility for delivering compounds and nanoparticles to sites of GBM induced blood brain barrier disruption. This novel strategy allows for targeting therapeutics via the underlying physiology of GBM rather than relying on cellular disease markers that are often lost in patients that relapse after targeting therapies.

This study demonstrates targeting of blood-brain barrier disruption to deliver payloads at sites of neurological disease., 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.

The blood–brain barrier (BBB) segregates the central nervous system from the systemic circulation. As such, the BBB not only prevents toxins and pathogens from entering the brain, but also limits the brain uptake of therapeutic molecules. However, under certain pathological conditions, the BBB is disrupted, allowing direct interaction between blood components and the diseased site. Moreover, techniques such as focused ultrasound can further disrupt the BBB in diseased regions. This review focuses on strategies that leverage such BBB disruption for delivering nanocarriers to the central nervous system (CNS). BBB disruption, as it relates to nanocarrier delivery, will be discussed in the context of acute pathologies such as stroke and traumatic brain injury, as well as chronic pathologies such as brain tumors, Alzheimer’s disease, and Parkinson’s disease. Key aspects of nanocarrier design as they relate to penetration and retention in the CNS are also highlighted.

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.

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.

INTRODUCTION The median survival of gliobastoma (GBM) patients remains less than two years despite aggressive treatments. Current targeted GBM therapies demonstrate initial therapeutic benefit; however, patients relapse due to therapeutic resistance and failure to eliminate GBM cells at the invasive margin. Therefore, we propose a two-prong approach: first, target pathologic disruption of the blood brain barrier (BBB) via exposure of neural ECM rather than disease markers to overcome therapy-resistant GBM; and second, designing therapeutic payloads that extracellularly spread throughout the tumor volume. METHODS Variable Lymphocyte Receptors (VLRs, a lamprey-derived antigen recognition system) were identified with high specificity for neural ECM. Candidate VLRs underwent further refinement using ex vivo tissue staining. Utilizing pathologic disruption of BBB as an approach for targeting GBM was confirmed in vivo with intracranial murine glioblastoma models. Finally, an immunogenic peptide was attached via a cleavable linker to the neural ECM binding VLRs for conditional release extracellularly to spread throughout the tumor. RESULTS The lead neural ECM-binding VLR candidate, named P1C10, demonstrates diffuse binding to parenchymal neural ECM, without detectable binding to other tissues. P1C10 demonstrates nanomolar affinity for neural ECM, and preferentially accumulates in intracranial GL261 and U87 murine GBM models. Finally, P1C10-targeted doxorubicin-loaded liposomes significant increased survival of mice with intracranial GBM. In additional studies, treating murine GBM models with a P1C10 VLR linked to an immunogenic peptide reduced GBM proliferation and increased infiltration of cytotoxic T cells. CONCLUSIONS We present proof-of-concept demonstration for targeting intracranial GBM via neural ECM exposed at pathological BBB disrupted sites. Additionally, P1C10 neural ECM-targeting VLR delivers chemotherapy-loaded nanoparticles and immunogenic peptides designed to spread extracellularly throughout the tumor. Thus, this novel strategy links a physiological ECM targeting scheme with extracellular-released therapeutics to treat primary GBM, and has potential for delivering therapies to other CNS diseases with pathological BBB.

INTRODUCTION: Median survival for newly diagnosed glioblastoma (GBM) patients remains less than two years despite aggressive clinical interventions. Antibody-based GBM-targeting could improve brain delivery and accumulation of therapies, but it has been challenging to identify universal tumor cell antigens due to intra-tumor and inter-patient GBM heterogeneity. We hypothesized that antigens of the neurovascular extracellular matrix (nvECM) within GBM-disrupted blood-brain barrier (BBB) may localize and enhance efficacy of therapeutic agents. METHODS: Yeast surface display biopanning and high throughput screening identified Variable Lymphocyte Receptors (VLR, i.e. jawless vertebrate lamprey antibodies) that preferentially accumulate at nvECM. Lead VLR candidates were tested for tumor targeting via combination with chemotherapeutic doxorubicin (DOX) liposomes and assessed for anti-GBM effects in vitro and against orthotopic mouse xenografts in vivo. RESULTS: VLR clones were enriched for binding to mouse brain endothelial cell (bEnd.3 line) ECM with two rounds of biopanning, then screened for preferential binding to brain-derived ECM. Lead VLR clone P1C10 was chosen for further testing for nvECM specificity vs. fibroblast (3T3 line) ECM in a high throughput screen, high affinity (Kd=48±6.1 nM), and immunolabeling of normal mouse and human brain, and GBM samples. Clone P1C10 demonstrated tumor-specific targeting in orthotopic U87 GBM xenografts, with 50% more DOX fluorescence detected at GBM after intravenous injection of P1C10-DOX liposomes compared to control, non-specific VLR-DOX. Finally, administration of P1C10-DOX liposomes (15 mg/kg, i.p., 1/week) significantly improved survival of mice bearing U87 xenografts, compared to non-targeted VLR-DOX (Kaplan-Meier, p CONCLUSIONS: We report combining yeast surface display biopanning and screening to identify VLRs that preferentially accumulate at disrupted nvECM of GBM-disrupted BBB, and demonstrate GBM-targeting specificity in vitro and in vivo with significant survival benefit. These results support the nvECM immune-targeting strategy to improve anti-GBM therapeutic efficacy, potentially improving survival and quality-of-life in brain cancer patients. Citation Format: Paul A. Clark, Benjamin J. Umlauf, Eric V. Shusta, John S. Kuo. Neurovascular-targeting antibodies discovered using yeast biopanning enhance drug delivery and improve survival for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1764.

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.