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. Author manuscript; available in PMC: 2015 Jun 30.
Published in final edited form as: Cancer Gene Ther. 2012 Mar 9;19(5):336–344. doi: 10.1038/cgt.2012.6

Intratumoral delivery of CD154 homolog (Ad-ISF35) induces tumor regression: analysis of vector biodistribution, persistence and gene expression

J Melo-Cardenas 1,3, M Urquiza 1,3, TJ Kipps 1,2, JE Castro 1,2
PMCID: PMC4486070  NIHMSID: NIHMS519133  PMID: 22402624

Abstract

Ad-ISF35 is an adenovirus (Ad) vector that encodes a mouse-human chimeric CD154. Ad-ISF35 induces activation of chronic lymphocytic leukemia (CLL) cells converting them into CLL cells capable of promoting immune recognition and anti-leukemia T-cell activation. Clinical trials in humans treated with Ad-ISF35-transduced leukemia cells or intranodal injection of Ad-ISF35 have shown objective clinical responses. To better understand the biology of Ad-ISF35 and to contribute to its clinical development, we preformed studies to evaluate biodistribution, persistence and toxicity of repeat dose intratumoral administration of Ad-ISF35 in a mouse model. Ad-ISF35 intratumoral administration induced tumor regression in more than 80% of mice bearing A20 tumors. There were no abnormalities in the serum chemistry. Mice receiving Ad-ISF35 presented severe extramedullary hematopoiesis and follicular hyperplasia in the spleen and extramedullary hematopoiesis with lymphoid hyperplasia in lymph nodes. After Ad-ISF35 injection, the vector was found primarily in the injected tumors with a biodistribution pattern that showed a rapid clearance with no evidence of Ad-ISF35 accumulation or persistence in the injected tumor or peripheral organs. Furthermore, pre-existing antibodies against Ad-5 did not abrogate Ad-ISF35 anti-tumor activity. In conclusion, intratumoral administration of Ad-ISF35 induced tumor regression in A20 tumor bearing mice without toxicities and with no evidence of vector accumulation or persistence.

Keywords: biodistribution, CD154, lymphoma

INTRODUCTION

CD40L, also known as CD154, is the main ligand for CD40. Signaling via CD40 activates antigen-presenting cells both in vitro and in vivo.1–3 CD40 ligation on dendritic cells induces cellular maturation and activation as detected by increased surface expression of co-stimulatory and major histocompatibility complex molecules, production of proinflammatory cytokines, such as interleukin 12, and enhanced T-cell activation.1 CD40 ligation of resting B cells increases antigen-presenting function and induces proliferation and immunoglobulin class switching.4 After CD154 stimulation, leukemia B cells become effective antigen-presenting cells and induce T-cell activation and cytotoxicity against leukemia cells. These effects are mediated by upregulation of co-stimulatory molecules (CD54, CD70, CD80 and CD86) and death receptors (CD95, DR5).5,6

Moreover, CD40/CD154 interaction may be a critical signaling component in the tumor microenvironment. It has been reported that the microenvironment can protect cancer cells from apoptosis and confer resistance to chemotherapy.7–9 These protected cancer cells can be the source of circulating tumor cells as well as metastatic tumor spread,10 and failure of current cancer treatments may be due, at least in part, to lack of activity in ‘highly protected’ niches of residual tumor cells embedded in the microenvironment. Recent data highlight the relevance of CD40-mediated immune activation of cells present in the microenvironment, specifically macrophages in which CD40 signaling can promote direct cytotoxicity that contributes to effective tumor surveillance.11

Interestingly, almost 100% of B-cell malignancies and up to 70% of solid tumors express CD40 and signaling through this pathway can result not only in cell activation, but also in apoptosis of cancer cells.12,13 Therefore, CD40/CD154 signaling can been considered a therapeutic target with applications not only in B-cell malignancy, but also in solid tumors.

Studies conducted with anti-CD40 antibodies or recombinant human CD40 ligand have reported objective clinical activity in patients with solid tumors, chronic lymphocytic leukemia (CLL) and lymphomas.11,14,15 In addition, our previous studies using adenovirus (Ad) vectors that encode mouse CD154 provide proof of principle showing that CD40/CD154-mediated immunotherapy is feasible and effective in cancer patients.15,16 However, patients who received autologous cells transduced to express mouse CD154 developed antibodies against it. Therefore, we have developed Ad-ISF35, which expresses a novel chimeric human-mouse CD40 binding protein that does not contain the antibody binding mouse domains targeted by human neutralizing antibodies (Nabs). ISF35 protein also resists cleavage, maximizing cell surface expression.

We have studied the safety of a single intratumoral injection of Ad-ISF35 in patients with chronic lymphocytic leukemia (CLL) (paper under review). In these studies, we have observed objective clinical responses with patients showing decreased leukemia cell counts as well as lymph node and spleen size reductions. Adverse events have been transient and mild, including ‘flu-like’ symptoms, hypophosphatemia and neutropenia.

On the basis of the encouraging results using a single Ad-ISF35 intranodal injection, we hypothesize that repeat dose administration will result in a greater clinical benefit. Therefore, we performed several studies to understand better the biology of Ad-ISF35 and to evaluate its safety, vector biodistribution, persistence and transgene expression after repeat dose intratumoral administration in a lymphoma mouse model using A20 cells implanted in BALB/c mice.

MATERIALS AND METHODS

Adenoviral vector

GMP quality Ad-ISF35 was provided by Memgen LLC (San Diego, CA) under licensing from University of California, San Diego (UCSD San Diego, CA). Ad-ISF35 was handled, purified and tested following GMP standards that satisfy Food and Drug Administration (FDA) requirements for clinical use in human patients. The vector was produced in 293 cells, which contained the E1 region of the viral genome to complement the deletion of that region from the vector itself. This deletion renders the vector incapable of replication outside the packaging cell line. We determined viral particles (vp per ml (mass spectrometry), infectious units (IU) per ml (Adeno-X rapid titer; Clontech, Mountain View, CA), presence of the ISF35 transgene (quantitative polymerase chain reaction (qPCR)), absence of endotoxins and replication-competent Ad, and sterility. This lot of vector passed all tests and each ml of Ad-ISF35 vector contained 4.46 × 1011 vp (7.69 × 1010 IU per ml). Recombinant Ad vector serotype 5 lacking the transgene was used as a control. Its production was performed using 293 cells and purified by cesium chloride gradients. Viral infection units were measured using Adeno-X rapid titer kit (Clontech) following the manufacturer’s instructions.

Animal inoculation

In all, 35 male and 35 female BALB/c mice, 6–7 weeks of age were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Explora Biolabs (San Diego, CA) performed in vivo animal studies. Biological samples were processed and tested at UCSD according to the biosafety guidelines and protocols from UCSD and in compliance with FDA guidelines.17 All animals were allowed to acclimate to the housing environment before implanting tumor cells. Animals were injected subcutaneously with 1 × 105 A20 cells (B-cell lymphoma cell line), in the right flank region. When average tumor size reached approximately 125–200 mm3, the animals were randomly divided into the groups (five males and five females per group) described in Table 1 to assure proper distribution of tumor size among the different groups. The animals were divided into groups and received single or repeat (three injections) administration of vehicle (Tris-lactose NaCl), 3 × 109 or 3 × 1010 vp of Ad-ISF35. Day 0 was defined as the day when mice received the first injection of vehicle or Ad-ISF35. Mice injected once were killed 5 days after Ad-ISF35 injection. The mice receiving repeat administration were killed 5 days after the last injection (19 days after the first injection). A separate group of animals receiving repeat administration of Ad-ISF35 (3 × 1010 vp) was followed up for 25 days (after the last injection) to evaluate for delayed toxicities.

Table 1.

Study design

Number of injections Euthanasia time point Treatment Dose (vp)
1 Day 5 Vehicle 0
Ad-ISF35 3 × 109
3 × 1010
3 Day 5 after third injection Vehicle 0
Ad-ISF35 3 × 109
3 × 1010
Follow-up Day 25 after third injection Ad-ISF35 3 × 1010
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Abbreviations: Ad, adenovirus; vp, viral particles.

Tumor size measurements

Tumor length and width were measured using a caliper. Tumor volumes were calculated by (length × width2)/2. If a second tumor occurred in a given mouse, both tumor volumes were measured and their volumes were added together.

Analysis of vector biodistribution

Vector biodistribution was analyzed in different organs and time points. First, we measured vector biodistribution after single administration of Ad-ISF35 (3 × 1010 vp) at 2, 6, 24, 48 and 120 h after injection. Second, we compared vector biodistribution at 120 h after single or repeat administration of Ad-ISF35. Third, we evaluated the effect of Nabs in mice pre-immunized with Ad-5 and Ad-ISF35 in vector biodistribution 2 h following a single Ad-ISF35 intratumoral administration. In all, 10 organs from each mouse (brain, lungs, heart, spleen, liver, kidney, small intestine, bone marrow, gonads and lymph nodes) and tumor tissues were harvested at the indicated time points and stored in RNALater (Valencia, CA) at −80 °C. Blood samples were not analyzed for vector levels because the volume taken from the animals was only sufficient to complete the blood chemistry and hematological tests.

Between 10 and 25 mg of each tissue were used for DNA isolation using the Qiagen kit (Valencia, CA) according to the manufacturer’s instructions. As bone marrow samples were formalin fixed, we followed Qiagen’s instructions for purification of genomic DNA (gDNA) from formalin-fixed tissues. DNA was quantified using the Nanodrop 1000 device and stored frozen at −20 °C.

RNA extraction was performed from tissue samples that had more than five copies of Ad-ISF35 DNA in 100 ng gDNA using Qiagen kit (Valencia, CA) following the manufacturer’s instructions. RNA was quantified by Nanodrop 1000 and stored frozen at −80 °C. In all, 100 ng of mRNA was converted to cDNA by reverse transcriptase PCR method for further analysis by qPCR.

Quantification of Ad-ISF35 vector and ISF35 gene expression

qPCR was performed using 100 ng of gDNA or cDNA per reaction. This assay was optimized to detect at least five DNA copies of ISF35 in 100 ng of gDNA or cDNA following FDA guidelines.17 All the samples were tested in duplicate with a cycle threshold (Ct) variability that was <5% for Ad-ISF35 amplification. The following primers at a concentration of 400 nM were used for ISF35 amplification: forward, 5′-CCTCTGGCTGAAGCCCAG-3′; reverse, 5′-CTCCCAAGTGAATGGATTGT-3′.

The TaqMan MGB-FAM probe 5′-TTACTCAAGGCGGCAAA-3′ was used at 250 nM. The PCR reaction was run using TaqMan qPCR Universal Master Mix w/UNG from Applied Biosystems (Foster City, CA). The qPCR program employed a denaturation step at 95 °C for 3 min. In all, 40 cycles of subsequent amplification steps were executed 95 °C for 20 s, 52 °C for 1 min and 20 s in Bio-Rad cycler iQ5 PCR machine. The expected size of the DNA fragment amplified by these primers was 100 bp. β-Actin gene was used as a positive control for DNA quality and PCR amplification. β-Actin gene amplification was conducted using the Applied Biosystems kit following the manufacturer’s instructions in 7900HT Fast Real-Time PCR system. β-Actin gene amplification was accomplished using the same PCR cycles’ temperature described above. To calculate the ISF35 DNA copies present in given sample of gDNA or cDNA, we followed the Applied Biosystems protocol.18

Clinical observations

The general health of the animals was monitored daily according to Explora BioLab’s standard operating procedures. Body weight and tumor volume were measured twice per week after Ad-ISF35 injection for a total of 4 weeks. Clinical observations were conducted and recorded twice per week beginning on the day of viral administration until the necropsy. The mice were euthanized at completion of the study according to the protocol and their organs (brain, lung, heart, spleen, liver, kidney, small intestine, gonads and lymph nodes) were harvested and weighed. Blood samples were collected via cardiocentesis under isoflurane anesthesia and animals were killed immediately by cervical dislocation. Blood samples were used for hematology and serum chemistry analysis and tissues were preserved for histopathology and molecular studies.

Hematology tests

A measure of 300 μl of blood was placed in a microcentrifuge tube containing EDTA and stored until tested. Hematology parameters analyzed included: white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cell differential, smear evaluation by technologist for white and red blood cell morphology and parasite screen, pathologist review of abnormal cells and absolute reticulocyte count.

Serum chemistry tests

A measure of 500 μl of blood was placed in serum separator tubes, allowed to clot at room temperature for 30–60 min and centrifuged at 10 000 g for 2 min. Serum was transferred into a fresh tube and placed on ice before being tested. Serum chemistry parameters analyses were: alkaline phosphatase, chloride, creatinine, glucose, phosphorous, alanine aminotransferase (SGPT-ALT), albumin, aspartate aminotransferase (SGOT-AST), total bilirubin, blood urea nitrogen, calcium, cholesterol, potassium, total protein and sodium.

Pathology analysis

Comprehensive necropsy was performed for each animal. Gross pathological observations were recorded. Selected organs (brain, lungs, heart, spleen, liver, kidney, small intestine, ovary or testis and lymph nodes) were harvested, weighted and macroscopic evaluations were performed and recorded.

Detection of Ad-Nabs assay

Mice were immunized subcutaneously with Ad-5 or Ad-ISF35 (3 × 1010 vp) once weekly for 3 weeks. Serum samples were collected 2 weeks after the last immunization. Neutralizing activity of the antibodies in the serum was assessed by pre-incubation of Ad-ISF35 (100 instruction for use) with serum samples at different dilutions (10−2–10−6) for 10 min at 37 °C. Then, the vector was added to 2 × 105 HeLa cells seeded in 96-well plates in a final volume of 100 μl. After 48 h of incubation, cells were harvested using trypsin-free solution and stained with anti-mCD154 antibody to detect ISF35 protein expression. Fluorescence data were acquired using FACSCalibur (BD Biosciences, San Diego, CA) and analyzed using Flow Jo v6.4.7 (Tree Star, Ashland, OR).

Statistical analysis

This study was conducted taking into account the minimum number of mice that allowed us to find a 10% difference between the treated and control groups with an α = 0.05 and power = 90% following the FDA guidelines.17

Group mean, standard deviation calculation and toxicology data were analyzed using one-way analysis of variance and the significance of intergroup differences was analyzed using Bonferroni’s ‘t’-test. The statistical analysis was performed using Prism Version 4.03, GraphPad Software (San Diego, CA).

RESULTS

Ad-ISF35 injections induced A20 tumor growth inhibition

Anti-tumor activity of Ad-ISF35 was analyzed in A20 tumor bearing mice receiving repeat administration of Ad-ISF35. Mice (female and male) were injected intratumorally with vehicle or Ad-ISF35 using 3 × 109 or 3 × 1010 vp per injection. All animals injected with vehicle showed tumor progression and were killed when tumors reached 2000 mm3 according to protocol guidelines. Neither a single administration of Ad-ISF35 nor vehicle resulted in tumor regression (Figure 1a). Mice receiving repeat administration of Ad-ISF35 showed inhibition of tumor growth and 81% of mice had complete remission of A20 lymphoma (P = 0.0005), with no significant difference among gender or vector dose injected (Figure 1b).

Figure 1.

Figure 1

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Ad-ISF35 intratumoral administration induces A20 tumor regression. Mice received a single (a) or repeat (b) intratumoral administration of vehicle or Ad-ISF35 using two different doses (3 × 109 or 3 × 1010 vp). The arrows indicate when the tumors were injected with Ad-ISF35. As a control, an empty adenovirus (Ad-5) was repeatedly administered using 3 × 1010 vp dose. (c) Photographs of mouse A20 lymphoma tumors 10 days after a single intratumoral injection with Ad-5 or Ad-ISF35.

Clinical observations

Mice were evaluated daily after A20 tumor development and Ad-ISF35 administration to identify toxicities and/or dead animals (n = 100). The rate of scheduled termination in this study was 95%. Five animals were found dead before the scheduled date of being killed. They showed massive tumor burden and multiple organ metastases. There were no differences in death frequency in the mice treated with vehicle control or Ad-ISF35. No additional micropathological review, vector biodistribution or transgene expression were performed for these animals due to tissue decomposition.

Mice that received Ad-ISF35 administration developed a hematoma and ecchymosis on the tumors 2–4 days after the first Ad-ISF35 administration (Figure 1c). We noticed that while the tumor size decreased, the initial hematoma turned into a dry scab. In addition, these mice developed mild-to-moderate rough coat on day 15. The frequency of rough coat was associated with the Ad-ISF35 injected dose, this was present in 50% of mice that received 3 × 109 vp and 100% of mice that received 3 × 1010 vp (data not shown). The control group that received vehicle only started to develop rapid tumor progression and necrosis on day 9 and did not form a hematoma-like bruise or dry scab tissue.

Effect of Ad-ISF35 on body and organ weights

Body and organ weights were measured in all animals on the scheduled termination date. In general, there were no significant changes in mean body weights of animals treated with single or repeat administration on either dose of Ad-ISF35. The lymph node weight was increased in the male and female groups that received single administration, but not in the group of mice treated with repeat administration of Ad-ISF35 compared with the vehicle group. The spleen weight was increased only in female mice receiving single administration with 3 × 1010 vp Ad-ISF35 compared with the vehicle group. Weight of reproductive organs was decreased in both male and female groups receiving vehicle compared with the mice treated with either dose of Ad-ISF35 (Supplementary Figure S1). The changes in organ weights were transient and did not persist for longer than 15 days after injection.

Effect of Ad-ISF35 on serum chemistry and hematology parameters

All of the laboratory parameters were found within normal range. However, there were statistical differences in neutrophils and platelet counts between the groups receiving single Ad-ISF35 administration and vehicle. Specifically, we observed increased neutrophil counts in females that received Ad-ISF35 with no difference among the dose groups, and increase in platelet counts in males receiving 3 × 1010 Ad-ISF35 (Supplementary Figure S2).

Histopathological findings

A comprehensive necropsy was conducted in all animals on the date of scheduled termination. There was no evidence of direct toxicity with Ad-ISF35 administration irrespective of the dose used. Brain, lungs, kidney, small intestine and ovary/testis tumor appeared to be normal. Histopathology analysis was only performed in mice treated with repeat administration of Ad-ISF35 and vehicle control (Table 2). Mice treated with Ad-ISF35 showed hepatic extramedullary hematopoiesis and appeared to have reduction in liver metastasis compared with vehicle control group, but this was not statistically significant. Only Ad-ISF35-treated mice developed lymphoid hyperplasia and extramedullary hematopoiesis in the lymph nodes (P = 0.0057). We observed extramedullary hematopoiesis and follicular hyperplasia in the spleen of mice injected with vehicle as well as Ad-ISF35. However, the presence of severe extramedullary hematopoiesis with follicular hyperplasia was more frequent in mice that received 3× 1010 vp Ad-ISF35 (P = 0.02).

Table 2.

Frequency of histopathology findings from mice treated with three injections of Ad-ISF35 and vehicle (0 vp)

Dose n Heart EC Lung M Liver
Spleen
Lymph nodes
M EMH EMH, FH Severe EMH FH Severe EMH, FH FH LH, EMH LH
0 vp 9 2 0 4 0 5 4 0 0 1 0 0
3 × 109 10 3 2 2 2 7 2 0 0 0 4 0
3 × 1010 10 2 0 2 3 2 3 1 4 1 2 4
Follow-up 3 × 1010 7 1 0 1 0 4 0 0 3 0 0 4
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Abbreviations: Ad, adenovirus; EC, epicardial calcification; EHM, extramedullary hematopoiesis; FH, follicular hyperplasia; LH, lymphoid hyperplasia; M, metastasis; vp, viral particles.

Epicardial calcification was found with similar frequency in mice that received Ad-ISF35 or vehicle. This is considered a nonspecific lesion commonly found in inbred mice.19

Overall, histopathology analysis of internal organs showed no evidence of direct toxic effects of Ad-ISF35 with any of the two doses used.

Ad-ISF35 DNA vector biodistribution after intratumoral administration

Vector biodistribution was analyzed at different time points (2, 6, 24, 48 and 120 h) after single administration of Ad-ISF35 at a fixed dose of 3 × 1010 vp. At 2 h after administration of Ad-ISF35, the vector was found in all 10 organs tested (copy number range 1 × 101 to 1 × 106 copies per 100 ng gDNA). Tumor tissues showed at least 1000 times higher copy number (~1 × 109 copies/100 ng gDNA) compared with the organs evaluated. We observed a rapid clearance of Ad-ISF35 vector from the organs tested during the first 24 h, with no evidence of persistence 5 days after intratumoral administration (Figure 2). In contrast, vector presence in tumor samples was still detectable at 5 days after administration with an Ad-ISF35 copy number of ~1 × 104 copies per 100 ng of gDNA.

Figure 2.

Figure 2

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Biodistribution of Ad-ISF35 after a single intratumoral administration. Mice were injected intratumorally with 3 × 1010 vp of Ad-ISF35 and peripheral organs were harvested at different time points as shown in the graph. Three mice were used at each time point. Using qPCR, vector presence was measured in 100 ng of gDNA with a limit of detection of 5 copies per 100 ng of gDNA as indicated by the dotted lines. The error bars represent mean±s.d.

We tested whether repeat administration of Ad-ISF35 (once every week for 3 weeks) impacts biodistribution and persistence of Ad-ISF35 vector. To assess this, we measured vector copy numbers in the injected tumor (using two doses 3 × 109 or 3 × 1010 vp) and peripheral organs 5 days after the third intratumoral administration of Ad-ISF35 and compared these values with those from mice that received only a single injection (Figure 3a and b).

Figure 3.

Figure 3

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Comparison of vector biodistribution after single or repeat dose administration of Ad-ISF35. (a) A20 tumor bearing mice received a single administration of Ad-ISF35 with two different doses and were killed 5 days after injection. Organs were harvested and tested for Ad-ISF35 DNA presence by qPCR. (b) Mice received repeat intranodal administration with Ad-ISF35 and organs were harvested 5 days after the third (last) injection. (c) Mice received vehicle control (0 vp). (d) A group of mice receiving Ad-ISF35 repeat administration (3 × 1010 vp) was followed for 25 days after the third injection to assess vector persistence. T, tumor; LN, lymph nodes; S, spleen; L, liver; K, kidney; BM, bone marrow; H, heart; Lu, lung; B, brain; G, gonads; I, intestine.

In general, we observed that injected tumors had higher copy numbers of Ad-ISF35 vector compared with peripheral organs. Very low copy numbers, close to the threshold of detection, were detected in lymph nodes from two mice treated with one injection (one in each dose group), in two spleen and two kidney samples from mice receiving multiple injections with 3 × 1010 vp of Ad-ISF35. Importantly, none of the mice evaluated showed transduction of liver cells with Ad-ISF35, this is reassuring as viral hepatitis with Ad vector can be the source of major clinical complications following Ad-based gene therapy. We also detected Ad-ISF35 in the testis of one mouse receiving one 3 × 1010 vp Ad-ISF35 injection (Figure 3a) and one mouse receiving vehicle (Figure 3c). These findings may be due to tissue contamination rather than true presence of Ad-ISF35 vector in the testicular tissue as it was also observed in the vehicle-treated mouse.

In tumor tissues, there was no significant statistical difference in Ad-ISF35 copy number in mice receiving single or repeat administration of Ad-ISF35 or the dose injected. Ad-ISF35 presence in tumors from mice receiving repeat administration (26%) was lower compared with mice receiving single administration (50%).

We examined Ad-ISF35 vector presence 25 days after the third injection with 3 × 1010 vp of Ad-ISF35 in a follow-up group of nine mice. Ad-ISF35 was detected in tumor tissue in one of seven samples (the other two mice had complete regression and no tumor tissue was found) with a copy number of 1.7 × 103 copies per 100 ng gDNA. Very low copy numbers (5–12 copies per 100 ng gDNA), close to the limit of detection (5 copies per 100 ng gDNA), were also detected in two spleen samples (Figure 3d).

Evaluation of ISF35 mRNA expression in tissues after intratumoral injection

We evaluated the mRNA expression of ISF35 (CD40 chimeric gene encoded by Ad-ISF35) in all positive samples for Ad-ISF35 DNA vector on day 5. Overall, 53% of samples positive for Ad-ISF35 DNA also showed ISF35 mRNA expression. ISF35 mRNA was found more frequently in mice receiving single (50%) vs repeat administration (17%). One kidney sample from a mouse receiving three injections of 3 × 1010 vp of Ad-ISF35 was positive for ISF35 mRNA. In the follow-up group, we observed ISF35 mRNA expression in the tumor tissue, but not in spleen samples (Table 3).

Table 3.

ISF35 mRNA expression in tissues positive for Ad-ISF35 DNA presence

Tissue ISF35 mRNA copy number per 100 ng cDNA
One injection
 3 × 109 Tumor 14
Tumor <5
Tumor NS
Tumor NS
Lymph nodes <5
 3 × 1010 Tumor 11
Tumor 68
Tumor 21
Tumor 19
Tumor <5
Tumor <5
Lymph nodes NS
Gonads <5
Three injections
 3 × 109 Tumor <5
 3 × 1010 Tumor <5
Tumor NS
Tumor NS
Spleen <5
Spleen <5
Kidney <5
Kidney 11
 Follow-up 3 × 1010 Tumor 40
Spleen <5
Spleen <5
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Abbreviations: Ad, adenovirus; NS, no sample was available because tumor tissue was very small (due to tumor regression) and enough only for DNA extraction.

Analysis of pre-existing Ad Nabs in anti-tumor activity induced by Ad-ISF35

We investigated the effect of pre-existing Nabs against Ad-5 or Ad-ISF35 on vector biodistribution and anti-tumor activity.

Mice were immunized by intraperitoneal injection of Ad-5 or Ad-ISF35 once every week for 3 weeks. Serum samples were evaluated for the presence of Nabs that could block Ad-ISF35 transduction of HeLa cells. All immunized mice showed Nabs titers higher than 1:10 000. There was no statistical difference between the antibody titers induced by Ad-5 or Ad-ISF35 or in their ability to neutralize Ad-ISF35 transduction of HeLa cells (Figure 4a). A20 cells were then implanted in the right flank of pre-immunized mice, and when tumors were >125 mm3, the mice received single Ad-ISF35 administration with 3 × 1010 vp. Mice were killed 2 h after Ad-ISF35 administration and vector presence in tumor tissues and organs was assessed by qPCR. Pre-existing antibodies against Ad-5 or Ad-ISF35 reduced vector distribution to most of the organs with a significant decrease of vector copy number in the injected tumor (Figure 4b).

Figure 4.

Figure 4

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Effect of neutralizing antibodies (Nabs) against Ad-5 or Ad-ISF35 in vector biodistribution and tumor regression. Nabs were induced in mice by intraperitoneal immunization using Ad-5 or Ad-ISF35. (a) Serum samples were collected and tested in vitro for their ability to neutralize Ad-ISF35 infection in HeLa cells. (b) Pre-immunized mice receiving a single Ad-ISF35 intratumoral injection (3 × 1010 vp) were killed 2 h later for detection of Ad-ISF35 DNA in tumor tissue and peripheral organs. (c) The in vivo activity of pre-existing anti-adenovirus Nabs was evaluated in mice previously immunized with Ad-5 (Imm Ad-5) or Ad-ISF35 (Imm Ad-ISF35) that were injected intratumorally with Ad-5 or Ad-ISF35. Tumor progression was measured over a period of 30 days.

We also measured in vivo the ability of Nabs against Ad-5 or Ad-ISF35 to block the activity of repeat intratumoral administration of Ad-ISF35. Our results showed that the anti-tumor activity of Ad-ISF35 was not abrogated by Nabs against Ad-5. On the contrary, Nabs against Ad-ISF35 completely blocked the anti-tumor activity of Ad-ISF35 (Figure 4c).

DISCUSSION

Ad-ISF35 is a replication-defective Ad encoding a genetically engineered, membrane-stabilized CD154 homolog (ISF35). CLL patients treated with autologous leukemic cells expressing ISF35 have shown clinical objective responses such as decrease in leukemia cell counts and decrease in the size of lymph nodes and spleen.15,16 However, this procedure requires leukapheresis of tumor cells, which are not always available and ex vivo manipulation of the cells at specialized facilities. Several groups have used intratumoral administration of recombinant adenoviral vectors as a promising strategy for effective local gene transfer,20–24 avoiding the hepatotoxicity and anaphylactic reactions induced by systemic intravenous administration.25–29 Therefore, we evaluated the administration of Ad-ISF35 by intratumoral direct injection, which allows us to expand potentially the clinical application of Ad-ISF35 in lymphomas and solid tumors.

We previously conducted preclinical studies in A20 tumor bearing mice in which intratumoral administration of Ad-ISF35, but not control vector or vehicle, resulted in tumor regression, systemic immune response and induction of protective immunity against subsequent challenge with A20 tumor cells (unpublished data). These encouraging results prompted us to perform a phase I clinical study in patients with CLL (paper submitted for publication). Patients in this trial received a single Ad-ISF35 administration directly into pathologically enlarged lymph nodes. We observed significant reductions in blood leukemia cell counts and a median reduction of 53% in the size of lymph nodes and/or spleen. Two out of 15 patients achieved a partial response based on the NCI-96 and iwCLL-2008 response criteria,30,31 and the response was durable (≥4 months) in nine patients. Although there was no evidence of Ad-ISF35 dissemination beyond the injected lymph node, the majority of peripheral blood leukemia cells expressed death receptors, pro-apoptotic proteins and immune co-stimulatory molecules. These phenotypic changes are similar to ‘bystander’ CLL cells co-cultured with Ad-ISF35 transduced cells in vitro (data not shown).

On the basis of these encouraging results, we hypothesize that repeat Ad-ISF35 administration could render a higher response rate. In preparation for these clinical trials in humans, we conducted experiments to study the safety, vector biodistribution, persistence, gene expression and effect of pre-existing antibodies against Ad after multiple Ad-ISF35 injections in A20 bearing BALB/c mice.

In general, all mice appeared to be healthy throughout the observation period (40 days). No major abnormalities were found by clinical observation, comprehensive necropsy, gross pathological observations, major organ weight, hematology, serum chemistry and histopathology analysis. It is interesting that mice injected with Ad-ISF35 had increases in platelet and neutrophil counts, which were also observed in our phase I intranodal study. Typically Ad-based therapies have been associated with thrombocytopenia.32–35 This observation suggests that Ad-ISF35 probably activates CD40 expressed on the platelet surface inducing platelet detachment from endothelial vessels or through other indirect mechanisms that can promote megakaryocyte maturation and/or platelet increase.36,37 Moreover, the fact that the platelet elevation has been consistently observed in mice and humans receiving Ad-ISF35 suggest that this laboratory parameter can be used as a surrogate marker for Ad-ISF35 systemic activity. The observed neutrophilia observed could be due to a direct interaction between Ad and neutrophils as shown before,38 or due to the release of cytokines after Ad administration.

In this mouse model, we show that repeat intratumoral administration of Ad-ISF35 induced significant tumor regression in the majority of mice regardless of the dose used (3–30 × 109 vp). One Ad-ISF35 injection as well as vehicle injection showed no anti-tumoral effect. Moreover, the activity of Ad-ISF35 was not due to the nonspecific effect from the Ad vector alone, as mice injected with Ad-5 (empty vector control) did not show tumor regression. After intratumoral injection, Ad-ISF35 DNA was found in all organs tested at 2 h. The vector copy number decreased rapidly after 6 h and by 24 h it was only detectable in tumor samples. This shows that Ad-ISF35 biodistribution after intratumoral injection in this animal model was broad with rapid vector clearance and lack of evidence for persistence or replication. Very importantly, Ad-ISF35 did not accumulate or infect cells in the liver or produce elevation in serum transaminases. These results seem to disagree with reports in the scientific literature that show hepatic dysfunction and Ad vector accumulation/persistence in the liver for more than 24 h after intratumoral Ad vector injection.23,39,40 The method of vector detection (qPCR vs luciferase chemoluminescence) or particular characteristics of the Ad-ISF35 vector may have influenced these results. Additional factors such as the tumor size, frequency, dose, volume of injections and vector degradation by the liver may influence organ distribution and persistence as well.41

Using a high-sensitivity qPCR assay, we found Ad-ISF35 DNA in the brain of mice immediately after intratumoral administration. The vector clearance from brain tissue was rapid and occurred within the first 6 h after Ad-ISF35 injection, suggesting that the source of the vector was circulating blood and not direct transduction of brain cells. In addition, analysis of brain tissues did not show histological abnormalities to suggest viral encephalitis or differences between Ad-ISF35-injected and vehicle-treated groups. Published reports typically had performed the assessment of vector biodistribution in the brain at 24 h or later time points and that is probably one of the reasons why they do not report similar findings.23,39

The dose of Ad-ISF35 did not have a major effect on vector biodistribution. We did not observe differences in organs from animals injected with either one of the doses used in our study (3 × 109 or 3 × 1010 vp). However, we observed a higher copy number in tumor tissues injected once with 3 × 1010 vp dose compared with the 3 × 109 vp dose, but the difference was not statistically significant. Repeat intratumoral administration of Ad-ISF35 had a lower frequency and copy number of positive tumors for Ad-ISF35 DNA and mRNA compared with single injected tumors. This observation might be related to the presence of Nabs against Ad-ISF35 or the decrease in the size of the target tumor injected, as mice experienced tumor regression with each Ad-ISF35 injection.

Histopathology analysis of mice treated with 3 × 1010 vp Ad-ISF35 showed severe extramedullary hematopoiesis and follicular hyperplasia in the spleen and lymphoid hyperplasia with extramedullary hematopoiesis in lymph nodes. This could be related to an immune response induced by the vector and/or the transgene (ISF35). Similar findings of severe splenic extramedullary hematopoiesis have been reported in mice receiving CpG oligodeoxynucleotides and it is believed these effects correspond to an immune/inflammatory-mediated process.42

The presence of pre-existing anti-Ad antibody titers could play a major role in the in vivo vector delivery and activity when injected into human tissues. Ad is a common virus that causes cold and the large majority of the population has anti-Ad antibodies with variable neutralizing activity.43–45 Therefore, we conducted experiments to study the role of neutralizing anti-Ad antibodies on the activity of Ad-ISF35 in vivo. The presence of pre-existing Nabs against Ad-5 did not abrogate the anti-tumor effect of Ad-ISF35 injections. The reason for this is not entirely clear. However, other reports have shown that antibodies against Ad-5 do not neutralize the activity of recombinant Ad vectors.46 It is possible that the presence of Nabs may contribute to dendritic cell activation, increasing anti-tumoral activity as shown previously.47 On the other hand, it has been reported that anti-tumor activity using CD154 can be achieved with only 0.3% of cells expressing CD154.48 All these factors could explain why intratumoral administration of Ad-ISF35 was still effective despite the presence of anti-Ad vector serotype 5 Nabs.

The observation that Nabs against Ad-ISF35 abrogated tumor response could be due to production of anti-ISF35 antibodies that interfered with its activity. Because ISF35 is an engineered human-mouse chimeric CD154 homolog in which the majority of the sequence is human (unpublished data; Memgen LLC), and the sites recognized by Nabs in previous clinical trials were removed, it is expected that this protein could be immunogenic in mice but much less, if at all, in humans. To investigate this issue, we plan to evaluate the production of anti-ISF35 antibodies in human patients enrolled in phase II clinical trials using Ad-ISF35. Taken together, these results suggest that naturally occurring anti-Ad antibodies may not reduce the activity of Ad-ISF35 when used in humans.

In conclusion, Ad-ISF35 intratumoral administration induced anti-tumoral activity with eradication of A20 lymphoma in more than 80% of mice, showed a safe administration profile and did not induce signs of toxicity in A20 bearing mice. Ad-ISF35 vector was rapidly cleared with no evidence of accumulation or persistence in this animal model. Ad-ISF35 anti-tumor activity was preserved even when Nabs against wild-type Ad (Ad-5) were present. The safe biodistribution profile, lack of vector persistence and activity of Ad-ISF35 provides the rationale to continue the clinical development of this vector with the aim to treat patients with B-cell malignancies and CD40 expressing solid tumors.

Supplementary Material

S Figure 1
S Figure 2

Acknowledgments

We thank Memgen LLC for providing Ad-ISF35 GMP quality; Explora Biolabs for performing the in vivo studies; and the Alliance for Cancer Gene Therapy (TJK, JEC, JMC, MU) and FDA-OOPD-R01-3427 Grant (JEC, TJK).

Footnotes

CONFLICT OF INTEREST

TJK receives stock options as a payment for providing consulting services to Memgen LLC. The University of California owns the patent for Ad-ISF35 and licenses it to Memgen LLC. The other authors declare no competing financial interests.

Supplementary Information accompanies the paper on Cancer Gene Therapy website (http://www.nature.com/cgt)

References

  • 1.Ma DY, Clark EA. The role of CD40 and CD154/CD40L in dendritic cells. Semin Immunol. 2009;21:265–272. doi: 10.1016/j.smim.2009.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Buhmann R, Nolte A, Westhaus D, Emmerich B, Hallek M. CD40-activated B-cell chronic lymphocytic leukemia cells for tumor immunotherapy: stimulation of allogeneic versus autologous T cells generates different types of effector cells. Blood. 1999;93:1992–2002. [PubMed] [Google Scholar]
  • 3.Serba S, Schmidt J, Wentzensen N, Ryschich E, Märten A. Transfection with CD40L induces tumour suppression by dendritic cell activation in an orthotopic mouse model of pancreatic adenocarcinoma. Gut. 2008;57:344–351. doi: 10.1136/gut.2007.130252. [DOI] [PubMed] [Google Scholar]
  • 4.Cerutti A, Puga I, Cols M. Innate control of B cell responses. Trends Immunol. 2011;32:202–211. doi: 10.1016/j.it.2011.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Kato K, Cantwell MJ, Sharma S, Kipps TJ. Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells. J Clin Invest. 1998;101:1133–1141. doi: 10.1172/JCI1472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chu P, Wierda WG, Kipps TJ. CD40 activation does not protect chronic lymphocytic leukemia B cells from apoptosis induced by cytotoxic T lymphocytes. Blood. 2000;95:3853–3858. [PubMed] [Google Scholar]
  • 7.Chantrain CF, Feron O, Marbaix E, DeClerck YA. Bone marrow microenvironment and tumor progression. Cancer Microenviron. 2008;1:23–35. doi: 10.1007/s12307-008-0010-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Mueller MM, Fusenig NE. Friends or foes-bipolar effects of the tumour stroma in cancer. Nat Rev Cancer. 2004;4:839–849. doi: 10.1038/nrc1477. [DOI] [PubMed] [Google Scholar]
  • 9.Joyce JA. Therapeutic targeting of the tumor microenvironment. Cancer Cell. 2005;7:513–520. doi: 10.1016/j.ccr.2005.05.024. [DOI] [PubMed] [Google Scholar]
  • 10.Herishanu Y, Pérez-Galán P, Liu D, Biancotto A, Pittaluga S, Vire B, et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood. 2011;117:563–574. doi: 10.1182/blood-2010-05-284984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Beatty GL, Chiorean EG, Fishman MP, Saboury B, Teitelbaum UR, Sun W, et al. CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans. Science. 2011;331:1612–1616. doi: 10.1126/science.1198443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Tong AW, Stone MJ. Prospects for CD40-directed experimental therapy of human cancer. Cancer Gene Ther. 2003;10:1–13. doi: 10.1038/sj.cgt.7700527. [DOI] [PubMed] [Google Scholar]
  • 13.Eliopoulos AG, Young LS. The role of the CD40 pathway in the pathogenesis and treatment of cancer. Curr Opin Pharmacol. 2004;4:360–367. doi: 10.1016/j.coph.2004.02.008. [DOI] [PubMed] [Google Scholar]
  • 14.Law CL, Grewal IS. Therapeutic interventions targeting CD40L (CD154) and CD40: the opportunities and challenges. Adv Exp Med Biol. 2009;647:8–36. doi: 10.1007/978-0-387-89520-8_2. [DOI] [PubMed] [Google Scholar]
  • 15.Wierda WG, Cantwell MJ, Woods SJ, Rassenti LZ, Prussak CE, Kipps TJ, et al. CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia. Blood. 2000;96:2917–2924. [PubMed] [Google Scholar]
  • 16.Wierda WG, Castro JE, Aguillon R, Sampath D, Jalayer A, McMannis J, et al. A phase I study of immune gene therapy for patients with CLL using a membrane-stable, humanized CD154. Leukemia. 2010;24:1893–1900. doi: 10.1038/leu.2010.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Services USDoHaH; Administration FaD, Research CfBEa, editor. B. Considerations for preclinical study design to assess vector biodistribution and persistence. Rockville, MD, USA: 2006. Guidance for Industry: Gene Therapy Clinical Trials-Observing Subjects for Delayed Adverse Events. [Google Scholar]
  • 18.Biosystems A. Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for use in Quantitative PCR. F Hoffmann-La Roche Ltd; Basel: [Google Scholar]
  • 19.Suttie AW. Histopathology of the spleen. Toxicol Pathol. 2006;34:466–503. doi: 10.1080/01926230600867750. [DOI] [PubMed] [Google Scholar]
  • 20.MacGill RS, Davis TA, Macko J, Mauceri HJ, Weichselbaum RR, King CR, et al. Local gene delivery of tumor necrosis factor alpha can impact primary tumor growth and metastases through a host-mediated response. Clin Exp Metastasis. 2007;24:521–531. doi: 10.1007/s10585-007-9089-3. [DOI] [PubMed] [Google Scholar]
  • 21.Kim JH, Lee YS, Kim H, Huang JH, Yoon AR, Yun CO. Relaxin expression from tumor-targeting adenoviruses and its intratumoral spread, apoptosis induction, and efficacy. J Natl Cancer Inst. 2006;98:1482–1493. doi: 10.1093/jnci/djj397. [DOI] [PubMed] [Google Scholar]
  • 22.Hu Z, Garen A. Intratumoral injection of adenoviral vectors encoding tumor-targeted immunoconjugates for cancer immunotherapy. Proc Natl Acad Sci USA. 2000;97:9221–9225. doi: 10.1073/pnas.97.16.9221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lohr F, Huang Q, Hu K, Dewhirst MW, Li CY. Systemic vector leakage and transgene expression by intratumorally injected recombinant adenovirus vectors. Clin Cancer Res. 2001;7:3625–3628. [PubMed] [Google Scholar]
  • 24.Reay J, Kim SH, Lockhart E, Kolls J, Robbins PD. Adenoviral-mediated, intratumor gene transfer of interleukin 23 induces a therapeutic antitumor response. Cancer Gene Ther. 2009;16:776–785. doi: 10.1038/cgt.2009.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Pande K, Ueda R, Machemer T, Sathe M, Tsai V, Brin E, et al. Cancer-induced expansion and activation of CD11b+ Gr-1+ cells predispose mice to adenoviral-triggered anaphylactoid-type reactions. Mol Ther. 2009;17:508–515. doi: 10.1038/mt.2008.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Oberholzer C, Oberholzer A, Tschoeke SK, Minter RM, Bahjat FR, LaFace D, et al. Influence of recombinant adenovirus on liver injury in endotoxicosis and its modulation by IL-10 expression. J Endotoxin Res. 2004;10:393–401. doi: 10.1179/096805104225005832. [DOI] [PubMed] [Google Scholar]
  • 27.Mahasreshti PJ, Kataram M, Wang MH, Stockard CR, Grizzle WE, Carey D, et al. Intravenous delivery of adenovirus-mediated soluble FLT-1 results in liver toxicity. Clin Cancer Res. 2003;9:2701–2710. [PubMed] [Google Scholar]
  • 28.Manickan E, Smith JS, Tian J, Eggerman TL, Lozier JN, Muller J, et al. Rapid Kupffer cell death after intravenous injection of adenovirus vectors. Mol Ther. 2006;13:108–117. doi: 10.1016/j.ymthe.2005.08.007. [DOI] [PubMed] [Google Scholar]
  • 29.Xu Z, Smith JS, Tian J, Byrnes AP. Induction of shock after intravenous injection of adenovirus vectors: a critical role for platelet-activating factor. Mol Ther. 2010;18:609–616. doi: 10.1038/mt.2009.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O’Brien S, et al. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood. 1996;87:4990–4997. [PubMed] [Google Scholar]
  • 31.Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008;111:5446–5456. doi: 10.1182/blood-2007-06-093906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lozier JN, Csako G, Mondoro TH, Krizek DM, Metzger ME, Costello R, et al. Toxicity of a first-generation adenoviral vector in rhesus macaques. Hum Gene Ther. 2002;13:113–124. doi: 10.1089/10430340152712665. [DOI] [PubMed] [Google Scholar]
  • 33.Eggerman TL, Mondoro TH, Lozier JN, Vostal JG. Adenoviral vectors do not induce, inhibit, or potentiate human platelet aggregation. Hum Gene Ther. 2002;13:125–128. doi: 10.1089/10430340152712674. [DOI] [PubMed] [Google Scholar]
  • 34.Wolins N, Lozier J, Eggerman TL, Jones E, Aguilar-Córdova E, Vostal JG. Intravenous administration of replication-incompetent adenovirus to rhesus monkeys induces thrombocytopenia by increasing in vivo platelet clearance. Br J Haematol. 2003;123:903–905. doi: 10.1046/j.1365-2141.2003.04719.x. [DOI] [PubMed] [Google Scholar]
  • 35.Cichon G, Schmidt HH, Benhidjeb T, Löser P, Ziemer S, Haas R, et al. Intravenous administration of recombinant adenoviruses causes thrombocytopenia, anemia and erythroblastosis in rabbits. J Gene Med. 1999;1:360–371. doi: 10.1002/(SICI)1521-2254(199909/10)1:5<360::AID-JGM54>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
  • 36.Inwald DP, McDowall A, Peters MJ, Callard RE, Klein NJ. CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation. Circ Res. 2003;92:1041–1048. doi: 10.1161/01.RES.0000070111.98158.6C. [DOI] [PubMed] [Google Scholar]
  • 37.Lievens D, Eijgelaar WJ, Biessen EA, Daemen MJ, Lutgens E. The multi-functionality of CD40L and its receptor CD40 in atherosclerosis. Thromb Haemost. 2009;102:206–214. doi: 10.1160/TH09-01-0029. [DOI] [PubMed] [Google Scholar]
  • 38.Cotter MJ, Zaiss AK, Muruve DA. Neutrophils interact with adenovirus vectors via Fc receptors and complement receptor 1. J Virol. 2005;79:14622–14631. doi: 10.1128/JVI.79.23.14622-14631.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wang Y, Yang Z, Liu S, Kon T, Krol A, Li CY, et al. Characterisation of systemic dissemination of nonreplicating adenoviral vectors from tumours in local gene delivery. Br J Cancer. 2005;92:1414–1420. doi: 10.1038/sj.bjc.6602494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Wang Y, Wang H, Li CY, Yuan F. Effects of rate, volume, and dose of intratumoral infusion on virus dissemination in local gene delivery. Mol Cancer Ther. 2006;5:362–366. doi: 10.1158/1535-7163.MCT-05-0266. [DOI] [PubMed] [Google Scholar]
  • 41.Bazan-Peregrino M, Carlisle RC, Purdie L, Purdie L, Seymour LW. Factors influencing retention of adenovirus within tumours following direct intratumoural injection. Gene Ther. 2008;15:688–694. doi: 10.1038/gt.2008.2. [DOI] [PubMed] [Google Scholar]
  • 42.Sparwasser T, Hültner L, Koch ES, Luz A, Lipford GB, Wagner H. Immunostimulatory CpG-oligodeoxynucleotides cause extramedullary murine hemopoiesis. J Immunol. 1999;162:2368–2374. [PubMed] [Google Scholar]
  • 43.Harvey BG, Hackett NR, El-Sawy T, Rosengart TK, Hirschowitz EA, Lieberman MD, et al. Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J Virol. 1999;73:6729–6742. doi: 10.1128/jvi.73.8.6729-6742.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Chen Y, Yu DC, Charlton D, Henderson DR. Pre-existent adenovirus antibody inhibits systemic toxicity and antitumor activity of CN706 in the nude mouse LNCaP xenograft model: implications and proposals for human therapy. Hum Gene Ther. 2000;11:1553–1567. doi: 10.1089/10430340050083289. [DOI] [PubMed] [Google Scholar]
  • 45.Chirmule N, Propert K, Magosin S, Qian Y, Qian R, Wilson J. Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther. 1999;6:1574–1583. doi: 10.1038/sj.gt.3300994. [DOI] [PubMed] [Google Scholar]
  • 46.Bramson JL, Hitt M, Gauldie J, Graham FL. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination. Gene Ther. 1997;4:1069–1076. doi: 10.1038/sj.gt.3300508. [DOI] [PubMed] [Google Scholar]
  • 47.Mercier S, Rouard H, Delfau-Larue MH, Eloit M. Specific antibodies modulate the interactions of adenovirus type 5 with dendritic cells. Virology. 2004;322:308–317. doi: 10.1016/j.virol.2004.01.031. [DOI] [PubMed] [Google Scholar]
  • 48.Grossmann ME, Brown MP, Brenner MK. Antitumor responses induced by transgenic expression of CD40 ligand. Hum Gene Ther. 1997;8:1935–1943. doi: 10.1089/hum.1997.8.16-1935. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

S Figure 1
NIHMS519133-supplement-S_Figure_1.pdf (1MB, pdf)
S Figure 2
NIHMS519133-supplement-S_Figure_2.pdf (607.9KB, pdf)

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