Sara D'Angelo

Ligand-targeted theranostic nanomedicines against cancer.

Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentially overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. The modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy.

The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.

From deep sequencing to actual clones.

The application of deep sequencing to in vitro display technologies has been invaluable for the straightforward analysis of enriched clones. After sequencing in vitro selected populations, clones are binned into identical or similar groups and ordered by abundance, allowing identification of those that are most enriched. However, the greatest strength of deep sequencing is also its greatest weakness: clones are easily identified by their DNA sequences, but are not physically available for testing without a laborious multistep process involving several rounds of polymerization chain reaction (PCR), assembly and cloning. Here, using the isolation of antibody genes from a phage and yeast display selection as an example, we show the power of a rapid and simple inverse PCR-based method to easily isolate clones identified by deep sequencing. Once primers have been received, clone isolation can be carried out in a single day, rather than two days. Furthermore the reduced number of PCRs required will reduce PCR mutations correspondingly. We have observed a 100% success rate in amplifying clones with an abundance as low as 0.5% in a polyclonal population. This approach allows us to obtain full-length clones even when an incomplete sequence is available, and greatly simplifies the subcloning process. Moreover, rarer, but functional clones missed by traditional screening can be easily isolated using this method, and the approach can be extended to any selected library (scFv, cDNA, libraries based on scaffold proteins) where a unique sequence signature for the desired clones of interest is available.

Integrated nanotechnology platform for tumor-targeted multimodal imaging and therapeutic cargo release

Significance The main goal in the emerging field of cancer nanomedicine is to generate, standardize, and produce multifunctional carriers designed to improve the response of drugs against tumors. Here we report the design, development, and preclinical validation of a ligand-directed bioinorganic platform that integrates tumor targeting, receptor-mediated cell internalization, photon-to-heat conversion, and drug delivery. This enabling hydrogel-based technology can accommodate a broad variety of ligands, nanoparticles, and payloads. We show experimental proof-of-concept in mouse models of breast and prostate cancer with molecular imaging and marked reduction of tumor growth. However, with future proof that this technology is translatable, medical applications beyond cancer may also be leveraged. A major challenge of targeted molecular imaging and drug delivery in cancer is establishing a functional combination of ligand-directed cargo with a triggered release system. Here we develop a hydrogel-based nanotechnology platform that integrates tumor targeting, photon-to-heat conversion, and triggered drug delivery within a single nanostructure to enable multimodal imaging and controlled release of therapeutic cargo. In proof-of-concept experiments, we show a broad range of ligand peptide-based applications with phage particles, heat-sensitive liposomes, or mesoporous silica nanoparticles that self-assemble into a hydrogel for tumor-targeted drug delivery. Because nanoparticles pack densely within the nanocarrier, their surface plasmon resonance shifts to near-infrared, thereby enabling a laser-mediated photothermal mechanism of cargo release. We demonstrate both noninvasive imaging and targeted drug delivery in preclinical mouse models of breast and prostate cancer. Finally, we applied mathematical modeling to predict and confirm tumor targeting and drug delivery. These results are meaningful steps toward the design and initial translation of an enabling nanotechnology platform with potential for broad clinical applications.

RECOMBINANT RENEWABLE POLYCLONAL ANTIBODIES

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

Profiling celiac disease antibody repertoire.

The aim of this study was to dissect the autoantibody response in celiac disease (CD) that remains largely unknown, with the goal of identifying the disease-specific autoantigenic protein pattern or the so called epitome. Sera from CD patients were used to select immunoreactive antigens from a cDNA phage-display library. Candidate genes were identified, the corresponding proteins produced and their immunoreactivity validated with sera from CD patients and controls. Thirteen CD-specific antigens were identified and further validated by protein microarray. The specificity for 6 of these antigens was confirmed by ELISA. Furthermore we showed that this antibody response was not abolished on a gluten free diet and was not shared with other autoimmune diseases. These antigens appear to be CD specific and independent of gluten induction. The utility of this panel extends beyond its diagnostic value and it may drive the attention to new targets for unbiased screens in autoimmunity research.

Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer

Significance Aggressive variant prostate cancer (AVPC) is a clinically defined tumor with neuroendocrine or small-cell differentiation, visceral metastases, low prostate-specific antigen, androgen receptor insensitivity, and poor/brief responses to androgen-deprivation or platinum-based chemotherapy. AVPC incidence has markedly increased, yielding an unmet diagnostic/therapeutic need. Here we adapted a patient-derived xenograft model and tumor samples to demonstrate ligand-directed theranostics of AVPC in vivo. We engineered human Herpes simplex virus thymidine kinase type-1 as a noninvasive imaging reporter/suicide transgene into adeno-associated virus/phage (AAVP) particles displaying motif ligands to cell surface-associated glucose-regulated protein 78kD (GRP78), toward a clinic-ready system. Although individual components of the AAVP system have been extensively investigated, this study is evidence of successful application in relevant preclinical models of untreatable and hard to diagnose aggressive tumor variants. Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC.

Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes

Significance Inflammatory breast cancer (IBC) is defined clinically and pathologically. Dermal lymphatic invasion is typical but is neither necessary nor sufficient for diagnosis; sentinel lymph node biopsy is contraindicated, challenging multidisciplinary management with upfront chemotherapy, surgery, and postoperative radiotherapy. Here we applied a ligand-directed “theranostic” (a combination of therapeutic and diagnostic) enabling platform to target IBC based on adeno-associated virus/phage (AAVP)-Herpes simplex virus thymidine kinase type-1 (HSVtk) particles displaying ligands to cell surface-associated 78-kD glucose-regulated protein (GRP78). In a suite of preclinical models and human tumor samples, we show simultaneous noninvasive molecular serial PET/CT imaging and targeted suicide transgene therapy. This study shows that a tumor-specific promoter, human GRP78 (hGRP78), can drive the expression of an imaging/suicide transgene in IBC and aggressive breast cancer in vivo. Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Specific binder for Lightning-Link® biotinylated proteins from an antibody phage library

Many applications required protein biotinylation. We routinely use biotinylated proteins to select single chain antibodies from phage and/or yeast display libraries. During phage selection the biotinylated antigens are bound to streptavidin coupled magnetic beads, while during yeast display, the biotinylated antigens are used during flow cytometry for both analysis and sorting. The Lightning-Link® Biotin kit, a rapid straightforward biotinylation kit that avoids the need for dialysis, is particularly useful when the amount of available protein is limiting. During routine screening of antibody libraries we identified a specific clone that bound a universal neo-epitope generated only when antigens are biotinylated with the commercial Lightning-Link® kit, with an affinity of ~10nM. Non-biotinylated proteins, and those biotinylated using alternative methods - the Thermo Fisher commercial kit or in vivo biotinylation using the Avitag (Ashraf et al., 2004) - were not recognized by this antibody. Using deep sequence analysis, the specific antibody was identified as being the most abundant in a number of different selections. This indicates the need for caution when using such modifying reagents, because of the possibility of selecting antibodies against the modification, rather than the target protein, and also highlights the value of deep sequencing analysis during display based selections. Furthermore, this antibody may have great utility in the analysis of proteins biotinylated using this method.

Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.