Delila Serra

Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines

CD4(+)CD25(high)FoxP3(+) regulatory T (Treg) cells limit antigen-specific immune responses and are a cause of suppressed anticancer immunity. In preclinical and clinical studies, we assessed the immune consequences of FoxP3(+) Treg-cell depletion in patients with advanced malignancies. We demonstrated that a CD25(high) targeting immunotoxin (denileukin diftitox) depleted FoxP3(+) Treg cells, decreased Treg-cell function, and enhanced antigen-specific T-cell responses in vitro. We then attempted to enhance antitumor immune responses in patients with carcinoembryonic antigen (CEA)-expressing malignancies by Treg-cell depletion. In a pilot study (n = 15), denileukin diftitox, given as a single dose or repeated dosing, was followed by immunizations with dendritic cells modified with the fowlpox vector rF-CEA(6D)-TRICOM. By flow cytometric analysis, we report the first direct evidence that circulating CD4(+)CD25(high)FoxP3(+) Treg cells are depleted after multiple doses of denileukin diftitox. Earlier induction of, and overall greater exposure to, the T-cell response to CEA was observed in the multiple-dose group, but not the single-dose group. These results indicate the potential for combining Treg-cell depletion with anticancer vaccines to enhance tumor antigen-specific immune responses and the need to explore dose and schedule of Treg depletion strategies in optimizing vaccine efforts.

Matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases

Insulin-like growth factor (IGF) bioavailability is modulated by specific IGFBPs, six of which are known (IGFBPs 1-6). Since IGFBPs have equal or higher affinity for IGFs than do IGF receptors, it is believed that degradation of IGFBPs by IGFBP-degrading proteinases is an important step in regulating IGF bioactivity. Recent studies from our laboratory have demonstrated that at least two IGFBPs, i.e. IGFBP-3 and -5, are degraded under physiologic conditions by matrix metalloproteinases (MMPs). In vitro, we have demonstrated that IGFBP-3 is degraded in human dermal fibroblast cultures by MMPs using a variety of techniques, including proteinase inhibition with a specific inhibitor of MMPs, i.e. tissue inhibitor of metalloproteinases (TIMP-1), immunoabsorption with specific antisera to human MMPs and a unique method developed in our laboratory, IGFBP-3 substrate zymography. Using similar methods, we have also demonstrated that MMPs, along with an unidentified 97-kDa proteinase, degrade IGFBP-5 in murine osteoblast cultures. In rat pregnancy serum, we have shown that degradation of IGFBP-3 is associated with MMP activity present in the serum, which likely arises from the placental compartment. Analysis of the cleavage products of IGFBP-3 produced by MMPs 1, 2 and 3 reveals that MMPs cleave exclusively within the non-homologous, mid-region of the molecule. Together, these studies suggest that MMPs, beyond their previously described roles as extracellular matrix degrading enzymes, may also exert effects on cellular growth and differentiation via degradation of IGFBPs.

Adenovirus Infection Triggers a Rapid, MyD88-Regulated Transcriptome Response Critical to Acute-Phase and Adaptive Immune Responses In Vivo

ABSTRACT Nearly 50 years ago, the discovery of interferon prompted the notion that host cells innately respond to viral invasion. Since that time, technological advances have allowed this response to be extensively characterized and dissected in vitro. However, these advances have only recently been applied to highly complex, in vivo biological systems. To this end, we exploited high-titer adenovirus (Ad) vectors to globally investigate the innate immune response to nonenveloped viral infection in vivo. Our results indicated a potent cellular transcriptome response shortly after infection, with global assessments revealing significant dysregulation in ∼15% of the measured transcripts derived from Ad vector-transduced tissue. Bioinformatics-based transcriptome analysis revealed a complex innate response to Ad infection, with induction of proinflammatory responses (and suppression of metabolism and mitochondrial genes) akin to those observed when mice are challenged with lipopolysaccharide. Despite this commonality, there were many unique aspects of the Ad-dependent transcriptome response, including the upregulation of several RNA regulatory mechanisms and apoptosis-related pathways, accompanied by the suppression of lysosomal and endocytic genes. Our results also implicated the Toll-like receptors (TLRs) in these responses, prompting specific investigations into this pathway. By using MyD88KO mice, our results confirmed that Ad-induced dysregulation of five functionally related gene clusters are significantly dependent on this TLR adaptor gene. MyD88 deficiency also resulted in significantly diminished, although not abolished, adaptive and acute-phase immune responses to Ad, confirming the transcriptome data, as well as specifically identifying MyD88 as a significant Ad immunity amplifier and regulator in vivo.

Liver Toxicities Typically Induced by First-Generation Adenoviral Vectors Can Be Reduced by Use of E1, E2b-Deleted Adenoviral Vectors

Adenoviral vectors from which the E1 region has been deleted ([E1(-)] Ad) are known to induce strong immune responses after systemic delivery. In this study we have evaluated liver toxicities in mice after intravenous injection with high doses of [E1(-)] or modified [E1(-), E2b(-)] Ad vectors (both expressing the bacterial beta-galactosidase [lacZ] marker gene) in C57BL/6, BALB/c, and SCID mice. Our data demonstrate a marked reduction in maximal liver toxicities and pathologies (typically noted at 21 days postinjection) with the use of the [E1(-), E2b(-)] modified vector in all strains of mice tested. Our data also demonstrated that despite the use of the [E1(-), E2b(-)] Ad vector, significant liver toxicities were still observed. To address this issue and the fact that the lacZ gene was perceived as a foreign antigen in the immune-competent C57BL/6 and BALB/c mice, we similarly injected mice tolerant of LacZ (lacZ-TG). In contrast to our studies in C57BL/6 and BALB/c mice, LacZ-TG mice exhibited virtually no evidence of hepatotoxicity after intravenous injection with the [E1(-), E2b(-)] vector, in contrast to use of the [E1(-)] Ad vector. Our results demonstrate that the [E1(-), E2b(-)] Ad vector class can reduce liver toxicities typically ascribed to Ad vector-mediated gene transfer after transfer of a highly immunogenic or foreign gene, whereas transfer of a transgene that is perceived as nonforeign by the host can be delivered with virtually no evidence of toxicity. On the basis of a careful review of the literature, these improvements in vector safety rival those noted with other, more significantly modified Ad vectors described to date.

Long-term efficacy after [E1-, polymerase-] adenovirus-mediated transfer of human acid-α-glucosidase gene into glycogen storage disease type II knockout mice

Glycogen storage disease type II (GSD-II) is a lethal, autosomal recessive metabolic myopathy caused by a lack of acid-alpha-glucosidase (GAA) activity in the cardiac and skeletal muscles. Absence of adequate intralysosomal GAA activity results in massive amounts of glycogen accumulation in multiple muscle groups, resulting in morbidity and mortality secondary to respiratory embarrassment and/or cardiomyopathy. In a mouse model of GSD-II, we demonstrate that infection of the murine liver with a modified adenovirus (Ad) vector encoding human GAA (hGAA) resulted in long-term persistence of the vector in liver tissues for at least 6 months. Despite both a rapid shutdown of hGAA mRNA expression from the vector, as well as the elicitation of anti-hGAA antibody responses (hGAA is a foreign antigen in this model), the hGAA secreted by the liver was taken up by all muscle groups analyzed and, remarkably, persisted in them for at least 6 months. The persistence of the protein also correlated with long-term correction of pathologic intramuscular glycogen accumulations in all muscle groups tested, but most notably the cardiac tissues, which demonstrated a significantly decreased glycogen content for at least 190 days after a single vector injection. The results suggest that gene therapy strategies may have the potential to significantly improve the clinical course for GSD-II patients.

Multiply deleted [E1, polymerase-, and pTP-] adenovirus vector persists despite deletion of the preterminal protein

The inherent limitations of [E1‐]Ad vectors as gene therapy vehicles suggest that further modifications may improve their overall performance profiles. However, Ad vector modifications can have untoward effects on their basic biology, e.g., some helper‐virus dependent Ad vectors have been found to be unstable without the presence of preterminal protein (pTP) activities. Despite this concern, we generated a new class of helper‐virus independent Ad vector that was multiply deleted for the E1, polymerase, and pTP genes, and investigated the ramifications of these deletions upon several vector performance parameters.

Adenovirus Vectors with the 100K Gene Deleted and Their Potential for Multiple Gene Therapy Applications

ABSTRACT The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K−]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1−,100K−]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K−]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K−]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1−,100K−]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K−]Ad vectors suggest that they may have utility in a variety of gene therapy applications.

Improved efficacy of gene therapy approaches for pompe disease using a new, immune-deficient GSD-II mouse model

Glycogen storage disease type II (GSD-II) is a lysosomal storage disorder in which the lack of human acid-alpha glucosidase (hGAA) activity results in massive accumulations of glycogen in cardiac and skeletal muscle fibers. Affected individuals die of cardiorespiratory failure secondary to the skeletal and/or cardiac muscle involvement. Recombinant hGAA enzyme replacement therapy (ERT) is currently in clinical trials and, although promising, ERT may be limited by large-scale production issues and/or the need for frequent infusions. These limitations could be circumvented or augmented by gene therapy strategies. Previous findings in our lab demonstrated that hepatic targeting of a modified adenovirus vector expressing human GAA was able to correct the glycogen accumulation in multiple affected muscles in the GAA-KO mice, by virtue of high-level, hepatic secretion of hGAA. However, although the vector persisted and expressed hGAA for 6 months in the liver, plasma hGAA was not detectable beyond 10 dpi (days postinjection), and reaccumulation of glycogen was observed. Two possibilities may have contributed to this phenomenon, the shut down of the CMV promoter and/or the onset of high levels of anti-hGAA antibodies. In order to test these and other possibilities, we have now developed an immune-deficient mouse model of GSD-II by interbreeding GAA-KO mice with severe combined immune-deficient (SCID) mice, generating double knockout, GAA-KO/SCID mice. In this new mouse model, we evaluated the efficacy of an [E1-, polymerase-] AdhGAA vector, in the absence of anti-hGAA antibody responses. After intravenous injection, GAA detection in the plasma was prolonged for at least 6 months secondary to the lack of anti-hGAA antibody production in all of the treated mice. GAA-KO/SCID mice treated with high doses of viral vector demonstrated longer durations of glycogen correction in both skeletal and cardiac muscles, relative to mice injected with lower doses of the vector. Notably, within 2 weeks of vector injection, muscle strength and coordination was normalized, and the improved muscle function persisted for at least 6 months. In summary, this new mouse model of GSD-II now makes it possible to assess the full potential for efficacy of any GAA-expressing vector (and/or ERT) contemplated for use in GSD-II gene therapy, without the negative influence that anti-hGAA antibodies entail.

Glycogen storage in multiple muscles of old GSD-II mice can be rapidly cleared after a single intravenous injection with a modified adenoviral vector expressing hGAA

Glycogen storage disease II (GSD‐II) is an autosomal recessive lysosomal storage disease, due to acid‐alpha‐glucosidase (GAA) deficiency. The disease is characterized by massive glycogen accumulation in the cardiac and skeletal muscles. There is early onset (infantile, also known as Pompe disease) as well as late onset (juvenile and adult) forms of GSD‐II. Few studies have been published to date that have explored the consequences of delivering a potential therapy to either late onset GSD‐II subjects, and/or early onset patients with long‐established muscle pathology. One recent report utilizing GAA‐KO mice transgenically expressing human GAA (hGAA) suggested that long‐established disease in both cardiac and skeletal muscle is likely to prove resistant to therapies. To investigate the potential for disease reversibility in old GSD‐II mice, we studied their responsiveness to exogenous hGAA exposure via a gene therapy approach that we have previously shown to be efficacious in young GAA‐KO mice.

Replication-attenuated Human Adenoviral Type 4 vectors elicit capsid dependent enhanced innate immune responses that are partially dependent upon interactions with the complement system

Human Adenovirus Type 4 (HAdV-4) is responsible for epidemic outbreaks of Acute Respiratory Disease (especially in military recruits), and is known to cause significant morbidity with several reported cases of mortality. However, we do not understand why this serotype causes such high morbidity, and have little insight into the immunobiology of HAdV-4 infections. We have now developed a replication attenuated HAdV-4 vector system, and through it, demonstrate that HAdV-4 virions have enhanced infectivity of certain cell types and reveal aspects of the serotype-specific heightened innate immunogenicity of infectious HAdV-4 capsids both in vitro and in vivo. We further found that elements of this serotype-specific immunogenicity were dependent upon interactions with the complement system. These findings provide insights into the mechanisms possibly underlying the known morbidity accompanying wild-type HAdV-4 infections as well as highlight important considerations when considering development of alternative serotype vectors.