Andrew P. Byrnes

Circulating Antibodies and Macrophages as Modulators of Adenovirus Pharmacology

ABSTRACT Adenovirus serotype 5 (Ad5) naturally infects the liver after intravenous injection, making it a candidate for hepatocyte-directed gene transfer. While Ad5 can be efficient, most of the dose is destroyed by liver Kupffer cells before it can reach hepatocytes. In contrast, Ad5 bearing the hexon from Ad6 (Ad5/6) evades Kupffer cells. While Ad5/6 dramatically increases hepatocyte transduction in BALB/c mice, it has surprisingly little effect on C57BL/6 mice. To determine the source of this strain-specific difference, the roles of Kupffer cells, liver sinusoidal endothelial cells (LSECs), hepatocytes, scavenger receptors, clotting factors, and immunoglobulins were analyzed. The numbers of Kupffer cells and LSECs, the level of clotting factor X, and hepatocyte infectibility did not differ between different strains of mice. In contrast, high levels of immunoglobulins correlated negatively with Ad5 liver transduction in different mouse strains. Removal of immunoglobulins by use of Rag-deficient mice restored Ad5 transduction to maximal levels. Removal of Kupffer cells by predosing or by testing in colony-stimulating factor knockout mice restored Ad5 transduction in the presence of immunoglobulins. Partial reconstitution of IgM in Rag mice resulted in significant reductions in liver transduction by Ad5 but not by Ad5/6. These data suggest a role for IgM-mediated clearance of Ad5 via Kupffer cells and may explain the mechanism by which Ad5/6 evades these cells. These mechanisms may play a vital role in Ad pharmacology in animals and in humans.

Induction of Shock After Intravenous Injection of Adenovirus Vectors: A Critical Role for Platelet-activating Factor

Innate immune responses are a major barrier to safe systemic gene therapy with adenovirus (Ad) vectors. We show that intravenous (IV) injection of rats with Ad5 vectors causes a novel rapid shock reaction that involves hypotension, hemoconcentration, tissue edema, and vasocongestion, with notable pathology in the pancreas and the gastrointestinal system. We show for the first time that this reaction is dependent on platelet-activating factor (PAF), a lipid signaling molecule that is a known shock inducer. Ad upregulated PAF within 5 minutes in vivo, and antagonists of the PAF receptor were able to prevent Ad-induced shock. Ad upregulated PAF via the reticuloendothelial system (RES), because splenectomy or depletion of phagocytes blocked the ability of Ad to induce both PAF and shock. Rats were considerably more sensitive to Ad-induced shock than were mice, but PAF mediated shock in both species. Other Ad-induced innate immune responses such as cytokine induction and thrombocytopenia were not mediated by PAF. In summary, systemic IV injection of Ad stimulates the RES to upregulate PAF within a matter of minutes, which results in shock. The identification of this novel pathway suggests strategies to improve the safety of systemic gene therapy with Ad vectors.

Impact of natural IgM concentration on gene therapy with adenovirus type 5 vectors

ABSTRACT Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice.

Substitution of blood coagulation factor X-binding to Ad5 by position-specific PEGylation: Preventing vector clearance and preserving infectivity

The biodistribution of adenovirus type 5 (Ad5) vector particles is heavily influenced by interaction of the particles with plasma proteins, including coagulation factor X (FX), which binds specifically to the major Ad5 capsid protein hexon. FX mediates hepatocyte transduction by intravenously-injected Ad5 vectors and shields vector particles from neutralization by natural antibodies and complement. In mice, mutant Ad5 vectors that are ablated for FX-binding become detargeted from hepatocytes, which is desirable for certain applications, but unfortunately such FX-nonbinding vectors also become sensitive to neutralization by mouse plasma proteins. To improve the properties of Ad5 vectors for systemic delivery, we developed a strategy to replace the natural FX shield by a site-specific chemical polyethylene glycol shield. Coupling of polyethylene glycol to a specific site in hexon hypervariable region 1 yielded vector particles that were protected from neutralization by natural antibodies and complement although they were unable to bind FX. These vector particles evaded macrophages in vitro and showed significantly improved pharmacokinetics and hepatocyte transduction in vivo. Thus, site-specific shielding of Ad5 vectors with polyethylene glycol rendered vectors FX-independent and greatly improved their properties for systemic gene therapy.

The role of endosomal escape and mitogen-activated protein kinases in adenoviral activation of the innate immune response.

Adenoviral vectors (AdV) activate multiple signaling pathways associated with innate immune responses, including mitogen-activated protein kinases (MAPKs). In this study, we investigated how systemically-injected AdVs activate two MAPK pathways (p38 and ERK) and the contribution of these kinases to AdV-induced cytokine and chemokine responses in mice. Mice were injected intravenously either with a helper-dependent Ad2 vector that does not express viral genes or transgenes, or with the Ad2 mutant ts1, which is defective in endosomal escape. We found that AdV induced rapid phosphorylation of p38 and ERK as well as a significant cytokine response, but ts1 failed to activate p38 or ERK and induced only a limited cytokine response. These results demonstrate that endosomal escape of virions is a critical step in the induction of these innate pathways and responses. We then examined the roles of p38 and ERK pathways in the innate cytokine response by administering specific kinase inhibitors to mice prior to AdV. The cytokine and chemokine response to AdV was only modestly suppressed by a p38 inhibitor, while an ERK inhibitor has mixed effects, lowering some cytokines and elevating others. Thus, even though p38 and ERK are rapidly activated after i.v. injection of AdV, cytokine and chemokine responses are mostly independent of these kinases.

Hexons from adenovirus serotypes 5 and 48 differentially protect adenovirus vectors from neutralization by mouse and human serum

Adenovirus vectors are widely used in gene therapy clinical trials, and preclinical studies with these vectors are often conducted in mice. It is therefore critical to understand whether mouse studies adequately predict the behavior of adenovirus vectors in humans. The most commonly-used adenovirus vectors are derived from adenovirus serotype 5 (Ad5). The Ad5 hexon protein can bind coagulation factor X (FX), and binding of FX has a major impact on vector interactions with other blood proteins. In mouse serum, FX protects Ad5 vectors from neutralization by natural antibodies and complement. In the current study, we similarly find that human FX inhibits neutralization of Ad5 vectors by human serum, and this finding is consistent among individual human sera. We show that human IgM and human IgG can each induce complement-mediated neutralization when Ad5 vectors are not protected by FX. Although mouse and human serum had similar effects on Ad5 vectors, we found that this was not true for a chimeric Ad5 vector that incorporated hexon regions from adenovirus serotype 48. Interestingly, this hexon-chimeric vector was neutralized by human serum, but not by mouse serum. These findings indicate that studies in mouse serum accurately predict the behavior of Ad5 vectors in human serum, but mouse serum is not an accurate model system for all adenovirus vectors.

Adenovirus Vector Toxicity

Adenovirus (Ad) vectors are one of the most commonly used classes of vectors being used in gene therapy clinical trials. However, vector-induced toxicity remains a significant barrier to safe, high-dose systemic therapy with Ad vectors. This review will describe what is known about the mechanisms of Ad-induced toxicity after administration of vector by the intravenous route, as well as how these toxicities can be mitigated. Given the hepatotropic nature of many commonly used Ad serotypes, the liver is a key site of virus-induced toxicity. Both innate and adaptive immunity contribute to hepatotoxicity. Intravenous delivery of Ad can also induce other rapid innate toxicities, including thrombocytopenia, systemic inflammation, fever and shock. Recent progress in understanding Ad biology has enabled improvements in vector safety and gene delivery efficiency in animal models, including genetic and chemical modification of the Ad vector itself, new ways to administer vector and pre-treatment with drugs that suppress innate and adaptive immune responses.

46. Shielding of Ad5 by Minimal Geneti-Chemical Modification: Preventing Clearance While Preserving Infectivity

The clinical efficacy of adenovirus serotype 5-based vectors is limited by complex interactions of the vector with host blood components. These interactions trigger sequestration of systemically administered vector particles mainly by the reticuloendothelial system. In order to generate retargeted Ad vectors suitable for i.v. delivery it is mandatory to understand and manipulate these non-target interactions.In mice, it was shown that blood coagulation factor X bound to hexon reduces sequestration by shielding the virus from natural antibodies and macrophage uptake, but also mediates hepatocyte transduction. To generate Ad vectors deficient for hepatocyte transduction but shielded from natural antibodies, we employed a combination of point mutations ablating (i) CAR binding (Ad-ΔCAR), (ii) integrin binding, (iii) FX binding (Ad-ΔFX), and rendered the vectors amenable for position-specific PEGylation at hexon HVR1 (Ad-HVR1) to compensate the lack of a FX-mediated shielding. Surface plasmon resonance analysis confirmed complete ablation of FX binding by ΔFX mutation in combination with PEGylation. Upon i.v. delivery, unPEGylated Ad-HVR1-ΔCAR-ΔFX did not transduce hepatocytes in BALB/c mice, whereas robust transduction levels were observed in antibody-deficient JHD mice. In agreement with the literature, this suggested that the ΔFX vector particles became susceptible to natural antibodies. However and surprisingly, PEGylated Ad-HVR1-ΔCAR-ΔFX showed a strong FX-independent hepatocyte transduction in both BALB/c and JHD mice compared to unPEGylated Ad-HVR1-ΔCAR-ΔFX and wildtype capsid vectors. Preliminary results suggested that integrins were involved in this FX-independent transduction. Further, PEGylated Ad-HVR1-ΔCAR-ΔFX vectors displayed 20-fold increased blood persistence without being associated to blood cells in BALB/c mice.To analyze vector sequestration by macrophages more closely we performed in vitro assays measuring the uptake of vector particles by Raw264.7 cells in the presence of murine plasma. We found enhanced uptake of unPEGylated Ad-HVR1-ΔFX vectors compared to wildtype capsid vectors. Using antibody-deficient and heated plasma of Ad naive mice confirmed a natural antibody- and complement-dependent uptake mechanism. This uptake was completely prevented by PEGylation of the vector particles, suggesting that position-specific PEGylation of hexon HVR1 interfered with natural antibody binding. Moreover, using hirudinized human whole blood we could show that a complement-dependent natural antibody-mediated binding of unPEGylated Ad-HVR1-ΔCAR-ΔFX vectors to erythrocytes could be prevented by PEGylation of hexon HVR1 with large PEG moieties.Thus, we conclude that a PEG-mediated evasion from sequestration by macrophages by shielding from natural antibodies or by a yet unknown mechanism maintained high vector concentrations in blood followed by enhanced hepatocyte transduction.

The FDA Review Process for Cancer Gene Therapy

This chapter provides an overview of the U.S. Food and Drug Administration (FDA) review process for cancer gene therapy products. Cancer gene therapy products are reviewed within FDA’s Center for Biologics Evaluation and Research (CBER). Although there are currently no FDA-approved gene therapies, CBER works with academic and industry sponsors during all stages of product development. CBER’s roles range from giving scientific advice to providing regulatory oversight to ensure the appropriate and safe treatment of human subjects. CBER teams composed of experts in various disciplines (chemistry/manufacturing/controls, pharmacology/toxicology, clinical, biostatistics, and regulatory project managers) review product and manufacturing issues, preclinical assessment, and clinical trial design at all stages of product development. This chapter summarizes CBER’s functions and highlights some issues that are of particular importance for sponsors of cancer gene therapy trials. References with further regulatory guidance and information are provided.