Matthew D. Stachler

Mosaic vectors comprised of modified AAV1 capsid proteins for efficient vector purification and targeting to vascular endothelial cells

Vascular-targeted gene therapies have the potential to treat many of the leading causes of mortality in the western world. Unfortunately, these therapies have been ineffective due to poor vascular gene transfer. The use of alternative virus serotypes and the incorporation of vascular targeting ligands into vectors has resulted in only modest increases in vascular gene transfer. Adeno-associated virus (AAV) 1 has shown the most promise among the AAV vectors for the transduction of vascular endothelial cells. However, no straightforward small-scale purification strategy exists for AAV1 as it does for AAV2 making it difficult to quickly produce AAV1 vector for analysis. Here we have combined two AAV1 capsid protein modifications to enhance vascular gene transfer and allow easy purification of vector particles. Mosaic vector particles have been produced comprised of capsid proteins containing the well-characterized RGD4C modification to target integrins present on the vasculature, and capsid proteins containing a modification that permits metabolic biotinylation and efficient purification of mosaic particles by avidin affinity chromatography. We show that the RGD modification results in a 50–100-fold enhancement in endothelial cell gene transfer that is maintained in biotinylated mosaic AAV1 particles. These results suggest that mosaic virions hold significant promise for targeted gene delivery to the vasculature.

Neonatal Fc Receptor Expression in Dendritic Cells Mediates Protective Immunity against Colorectal Cancer

Cancers arising in mucosal tissues account for a disproportionately large fraction of malignancies. Immunoglobulin G (IgG) and the neonatal Fc receptor for IgG (FcRn) have an important function in the mucosal immune system that we have now shown extends to the induction of CD8(+) T cell-mediated antitumor immunity. We demonstrate that FcRn within dendritic cells (DCs) was critical for homeostatic activation of mucosal CD8(+) T cells that drove protection against the development of colorectal cancers and lung metastases. FcRn-mediated tumor protection was driven by DCs activation of endogenous tumor-reactive CD8(+) T cells via the cross-presentation of IgG complexed antigens (IgG IC), as well as the induction of cytotoxicity-promoting cytokine secretion, particularly interleukin-12, both of which were independently triggered by the FcRn-IgG IC interaction in murine and human DCs. FcRn thus has a primary role within mucosal tissues in activating local immune responses that are critical for priming efficient anti-tumor immunosurveillance.

Site-specific modification of AAV vector particles with biophysical probes and targeting ligands using biotin ligase.

We have developed a highly specific and robust new method for labeling adeno-associated virus (AAV) vector particles with either biophysical probes or targeting ligands. Our approach uses the Escherichia coli enzyme biotin ligase (BirA), which ligates biotin to a 15-amino-acid biotin acceptor peptide (BAP) in a sequence-specific manner. In this study we demonstrate that by using a ketone isotere of biotin as a cofactor we can ligate this probe to BAP-modified AAV capsids. Because ketones are absent from AAV, BAP-modified AAV particles can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage modification methodology in the context of a mammalian cell lysate for the labeling of AAV vector particles with various fluorophores, and for the attachment of a synthetic cyclic arginine-glycine-aspartate (RGD) peptide (c(RGDfC)) to target integrin receptors that are present on neovasculature. Fluorophore labeling allowed the straightforward determination of intracellular particle distribution. Ligand conjugation mediated a significant increase in the transduction of endothelial cells in vitro, and permitted the intravascular targeting of AAV vectors to tumor-associated vasculature in vivo. These results suggest that this approach holds significant promise for future studies aimed at understanding and modifying AAV vector-cellular interactions.

Induction of HIV-1–Specific Mucosal Immune Responses Following Intramuscular Recombinant Adenovirus Serotype 26 HIV-1 Vaccination of Humans

BACKGROUND Defining mucosal immune responses and inflammation to candidate human immunodeficiency virus type 1 (HIV-1) vaccines represents a current research priority for the HIV-1 vaccine field. In particular, it is unclear whether intramuscular immunization can elicit immune responses at mucosal surfaces in humans. METHODS In this double-blind, randomized, placebo-controlled clinical trial, we evaluated systemic and mucosal immune responses to a candidate adenovirus serotype 26 (Ad26) vectored HIV-1 envelop (Env) vaccine in baseline Ad26-seronegative and Ad26-seropositive healthy volunteers. Systematic mucosal sampling with rectal Weck-Cel sponges and rectal biopsies were performed. RESULTS Intramuscular immunization elicited both systemic and mucosal Env-specific humoral and cellular immune responses in the majority of subjects. Individuals with preexisting Ad26-specific neutralizing antibodies had vaccine-elicited immune responses comparable to those of subjects who were Ad26 seronegative. We also observed no increase in activated total or vector-specific mucosal CD4+ T lymphocytes following vaccination by either histopathology or flow cytometry. CONCLUSIONS These data demonstrate that a single intramuscular administration of this Ad26-vectored HIV-1 Env vaccine elicited both systemic and mucosal immune responses in humans. Induction of antigen-specific humoral and cellular mucosal immunity was not accompanied by a detectable increase in mucosal inflammation. CLINICAL TRIALS REGISTRATION NCT01103687.

Epithelial PD-L2 Expression Marks Barrett's Esophagus and Esophageal Adenocarcinoma

Esophageal adenocarcinoma and Barretts esophagus epithelial cells commonly express PD-L2 in the setting of a Th2-skewed chronic inflammatory environment. Additional tumors express PD-L1 in immune cells. Evaluation of PD-1 inhibition and PD-L2 as biomarkers is warranted. Esophageal adenocarcinoma is an increasingly common disease with a dismal 5-year survival rate of 10% to 15%. In the first systematic evaluation of the PD-1 pathway in esophageal adenocarcinoma, we identify expression of PD-L2 in cancer cells in 51.7% of esophageal adenocarcinomas. Epithelial PD-L1 was expressed on only 2% of cases, although PD-L1+ immune cells were observed in 18% of esophageal adenocarcinomas. We also evaluated expression in the precursor lesion of esophageal adenocarcinoma, Barretts esophagus, which emerges following gastric reflux–induced esophageal inflammation, and found PD-L2 expression in Barretts esophagus but not in non–Barretts esophagus esophagitis. Because the progression from squamous esophagitis to Barretts esophagus is accompanied by a transition from a TH1 to TH2 immune response, we hypothesized that the TH2 cytokines IL4/IL13 could contribute to PD-L2 induction. We confirmed that these cytokines can augment PD-L2 expression in esophageal adenocarcinoma cell lines. These results suggest that the inflammatory environment in Barretts esophagus and esophageal adenocarcinoma may contribute to the expression of PD-L2. Furthermore, the potential for PD-1 receptor blockade to be effective in esophageal adenocarcinomas with epithelial PD-L2 or immune cell PD-L1 expression should be evaluated in clinical trials. Cancer Immunol Res; 3(10); 1123–9. ©2015 AACR.

Critical role for IL-1β in DNA damage-induced mucositis

Significance Deletion of the E3 β-TrCP in the mouse gut epithelium deregulates enterocyte cell cycle, induces a DNA damage response (DDR), and abolishes the epithelium barrier function, resulting in a lethal mucosal inflammation. Epithelial-derived IL-1β, likely induced by DDR independently of NF-κB, is a major culprit, and initiates the pathology by compromising epithelial tight junctions (TJs). Anti–IL-1β treatment secures the TJs and prevents the fulminant mucosal inflammation. IL-1β secretion accompanies human mucositis, a severe mucosal inflammatory reaction caused by chemoradiation therapy-induced DNA damage, which often results in treatment suspension. We propose that anti–IL-1β preventive treatment may ameliorate mucositis, as well as multiple disorders associated with epithelial barrier permeability, including burn injuries, head and neck trauma, alcoholic intoxication, and graft-vs.-host disease. β-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-κB inhibitor IκB. To appreciate tissue-specific roles of β-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1β as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1β is induced by DNA damage via an NF-κB–independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-κB, with failure to express the endogenous IL-1β receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1β prevents epithelial tight junction dysfunction and alleviates mucositis in β-TrCP–deficient mice. IL-1β antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.

Genomic Heterogeneity as a Barrier to Precision Medicine in Gastroesophageal Adenocarcinoma

Gastroesophageal adenocarcinoma (GEA) is a lethal disease where targeted therapies, even when guided by genomic biomarkers, have had limited efficacy. A potential reason for the failure of such therapies is that genomic profiling results could commonly differ between the primary and metastatic tumors. To evaluate genomic heterogeneity, we sequenced paired primary GEA and synchronous metastatic lesions across multiple cohorts, finding extensive differences in genomic alterations, including discrepancies in potentially clinically relevant alterations. Multiregion sequencing showed significant discrepancy within the primary tumor (PT) and between the PT and disseminated disease, with oncogene amplification profiles commonly discordant. In addition, a pilot analysis of cell-free DNA (cfDNA) sequencing demonstrated the feasibility of detecting genomic amplifications not detected in PT sampling. Lastly, we profiled paired primary tumors, metastatic tumors, and cfDNA from patients enrolled in the personalized antibodies for GEA (PANGEA) trial of targeted therapies in GEA and found that genomic biomarkers were recurrently discrepant between the PT and untreated metastases. Divergent primary and metastatic tissue profiling led to treatment reassignment in 32% (9/28) of patients. In discordant primary and metastatic lesions, we found 87.5% concordance for targetable alterations in metastatic tissue and cfDNA, suggesting the potential for cfDNA profiling to enhance selection of therapy.Significance: We demonstrate frequent baseline heterogeneity in targetable genomic alterations in GEA, indicating that current tissue sampling practices for biomarker testing do not effectively guide precision medicine in this disease and that routine profiling of metastatic lesions and/or cfDNA should be systematically evaluated. Cancer Discov; 8(1); 37-48. ©2017 AACR.See related commentary by Sundar and Tan, p. 14See related article by Janjigian et al., p. 49This article is highlighted in the In This Issue feature, p. 1.

Genetic and Epigenetic Alterations in Barrett’s Esophagus and Esophageal Adenocarcinoma

Esophageal adenocarcinoma (EAC) develops from Barretts esophagus (BE), wherein normal squamous epithelia is replaced by specialized intestinal metaplasia in response to chronic gastroesophageal acid reflux. BE can progress to low- and high-grade dysplasia, intramucosal, and invasive carcinoma. Both BE and EAC are characterized by loss of heterozygosity, aneuploidy, specific genetic mutations, and clonal diversity. Given the limitations of histopathology, genomic and epigenomic analyses may improve the precision of risk stratification. Assays to detect molecular alterations associated with neoplastic progression could be used to improve the pathologic assessment of BE/EAC and to select high-risk patients for more intensive surveillance.

Novel molecular insights from routine genotyping of colorectal carcinomas.

Routine tumor genotyping enables identification of concurrent mutations in tumors and reveals low-frequency mutations that may be associated with a particular tumor phenotype. We genotyped 311 colorectal carcinomas (CRCs) for 471 mutation hot spots in 41 cancer-associated genes. At least 1 mutation was present in 239 (77%) of 311 tumors. Two concurrent mutations were identified in 89 (29%) tumors, 3 mutations in 24 (8%), 4 mutations in 6 (2%), and 5 mutations in 1 tumor. KRAS mutations were most frequent and identified in 132 (42%) tumors, followed by APC in 79 (25%) and TP53 in 64 (21%) tumors. Mutations in PIK3CA, BRAF, CTNNB1, and NRAS were identified in 41, 27, 11, and 9 cases, respectively. Rare mutations not typically associated with CRC included AKT1 (4), AKT2 (1), IDH1 (1), KIT (1), MAP2K1 (1), PTEN (2), and GNAS (6). GNAS mutations in CRC correlated with a mucinous phenotype and were present in 20% of all mucinous adenocarcinomas evaluated in this study. Among CRCs with a PIK3CA mutation, 77% showed concurrent mutations in other cancer-associated genes, and 4% of CRC did not neatly fit into either the chromosomal instability pathway or CpG island methylator phenotype/microsatellite instability pathway, suggesting overlapping mutational profile in some tumors. Our findings indicate that routine tumor genotyping is helpful in identifying low-frequency mutations, such as GNAS, that may correlate with a specific morphological phenotype and also reveal multiplicity of concurrent mutations in a significant proportion of CRC that may have significant implications for clinical trial design and personalized therapy.

CDK4/6 or MAPK blockade enhances efficacy of EGFR inhibition in oesophageal squamous cell carcinoma

Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial–mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically.