Phase I Trial of a Pathotropic Retroviral Vector Expressing a Cytocidal Cyclin G1 Construct (Rexin-G) in Patients With Advanced Pancreatic Cancer

Abstract

Rexin-G is a pathotropic retroviral vector displaying a von Willebrand factor–targeting motif and expressing a dominant negative cyclin G1 gene. We undertook a phase I trial of intravenous (IV) administration of Rexin-G in patients with gemcitabine refractory, metastatic pancreatic adenocarcinoma. Twelve patients were treated. Dose escalation was performed from a dose of 1 × 10¹¹ colony forming units (CFU) per cycle to 6 × 10¹¹ CFU per cycle. The treatment was well tolerated. One dose-limiting toxicity (DLT) at dose level 2 (1.5 × 10¹¹ CFU per cycle) was observed, consisting of grade 3 transaminitis. There was no detection of replication-competent virus in patients’ peripheral blood mononuclear cells (PBMCs) or viral integration in DNA obtained from PBMCs, and no development of neutralizing antibodies. No evidence of antitumor activity was observed. The best objective response was progressive disease in 11 of the 12 study patients, while 1 patient showed radiographically stable disease with clinical deterioration and increase in the CA19.9 tumor marker. Median time to progression was 32 days. The median duration of survival of the study patients was 3.5 months from treatment initiation. Rexin-G is well tolerated in doses up to 6 × 10¹¹ CFU in patients with recurrent pancreatic cancer, but there was no evidence of clinical antitumor activity.

Introduction

Recurrent pancreatic cancer is a lethal disease with a dismal median survival of ≤6 months. The standard of care in this setting is gemcitabine chemotherapy, with or without the addition of erlotinib.¹ When patients fail gemcitabine, there is no intervention that can reliably extend survival. Novel therapeutic approaches are urgently needed.

Rexin-G is a nonreplicating retroviral vector deriving from the Moloney murine leukemia virus (MoMLV) family, displaying a von Willebrand factor–derived collagen-binding motif at its amphotropic envelope.² The virus encodes a mutant human cyclin G1 gene, incorporating a deletion at the region responsible for cyclin G1-CDK association and activation.^3,4 Blocking cyclin G1 activity with an antisense or mutant G1 construct led to inhibition of proliferation of pancreatic cancer cells,⁵ and portal vein administration of Rexin-G vector resulted in antitumor activity in a nude mouse model of pancreatic liver metastasis.³

On the basis of these preclinical data and initial human data from the Philippines, indicating encouraging duration of disease control in three patients with metastatic pancreatic cancer,⁶ we initiated a phase I/II trial of Rexin-G in patients with locally advanced or metastatic pancreatic cancer, refractory to gemcitabine. The goals of this trial were (i) to determine the dose-limiting toxicity (DLT) and maximum tolerated dose of Rexin-G administered as intravenous (IV) infusions in a dose range that matched or exceeded doses previously associated with antitumor activity in humans, (ii) to evaluate the viral kinetics of IV infusion of Rexin-G and its potential for invoking an immune response, recombination events, and vector integration in nontarget organs, and (iii) to assess antitumor activity of IV administered Rexin-G.

Results

Patient characteristics

Table 1 summarizes the baseline characteristics of the patients. Twelve patients with pancreatic adenocarcinoma were treated at three dose levels. Three patients were treated at dose level 1, six at dose level 2, and three at dose level 3. The patients comprised three women and nine men, with a median age of 60.5 years (range: 42–71 years). All the patients had metastatic disease at the time of entering this study, and in all of them gemcitabine chemotherapy had failed. The median number of prior chemotherapy regimens was 1 (range 1–3).

Table 1.

Patient characteristics

	Total (N = 12)
Age
Median	60.5
Range	(42–71)
Gender
F	3 (25%)
M	9 (75%)
Disease stage
Metastatic	12 (100%)
Performance score
0	5 (41.7%)
1	7 (58.3%)

Treatment schedule

Dose level 1 was defined as a dose of 7.5 × 10⁹ colony forming units (CFU) administered on days 1–7 and 15–21 of a one-cycle treatment course (total dose per cycle 1 × 10¹¹ CFU). All three of the patients enrolled at dose level 1 completed the entire therapy and received the full dose of Rexin-G. Dose level 2 was defined as a dose of 1.1 × 10¹⁰ CFU given on days 1–7 and 15–21 of a one-cycle treatment course (total dose per cycle 1.5 × 10¹¹ CFU). Six patients were treated at dose level 2, four of whom received the full dose of Rexin-G. The dose was reduced in one patient who developed grade 3 aspartate aminotransferase and alanine aminotransferase (which met the DLT definition in the protocol), and was withheld for 1 day in another patient who developed a reversible, grade 2 alkaline phosphatase elevation. Dose level 3 was defined as a per-day dose of 3.0 × 10¹⁰ CFU, to be administered over two cycles separated by 6 weeks. In each cycle, Rexin-G was given on days 1–5, 8–12, 15–19, 22–26 (total dose per cycle, 6 × 10¹¹ CFU). All three of the patients enrolled in this dose level completed the full dose for cycle 1, but none of the patients received cycle 2 treatment because of disease progression.

Toxicity

Toxicity was assessed using Common Terminology Criteria Version 3.0. Figure 1 summarizes treatment-related toxicities overall. Table 2 includes treatment-related toxicities by dose level. Toxicities include any adverse event that was attributed to be possibly, probably, or definitely related to the treatment.

Figure 1. Highest grade treatment-related toxicity per patient.

Most of the observed toxicity was grade 1. One dose-limiting toxicity was observed and consisted of grade 3 aspartate aminotransferase (AST) and alanine aminotransferase (ALT) elevation. ABD distention, abdominal distention; ALK phosphatase, alkaline phosphatase; ANC, absolute neutrophil count; serum glutamic-oxalocetic transaminase; SGPT, serum glutamic-pyruvic transaminase.

Table 2.

Rexin-G treatment–related toxicity according to dose level

Dose level	Toxicity	Grade
Dose level 1	Anorexia	2
	Flushing	1
	Nausea	1
	Fever-no ANC	1
	Abdominal distention	2
	Insomnia	1
	Diarrhea	1
Dose level 2	Hypermagnesemia	1
	SGPT (ALT)	3
	SGOT (AST)	3
	ALK phosphatase	1
Dose level 3	Diarrhea	1
	Nausea	1

Abbreviations: ALT, alanine aminotransferase; ANC, absolute neutrophil count; AST, aspartate aminotransferase; SGOT, serum glutamic-oxalocetic transaminase; SGPT, serum glutamic-pyruvic transaminase.

At dose level 1, all three patients were evaluated for toxicity, and none of them experienced a DLT. Two patients at this dose level experienced treatment-related anorexia (grades 1 and 2).

At dose level 2, six eligible patients were evaluated for toxicity. One patient had dose-limiting toxicities. This patient experienced a grade 3 aspartate aminotransferase and a grade 3 alanine aminotransferase elevation, which were both considered to be possibly related to the treatment. The treatment was withheld, and the transaminase elevation reversed to grade 1 in 96 hours. Because this patient had been enrolled in the first cohort of three patients at dose level 2, an additional cohort of three patients was enrolled to this dose level, and in these patients no DLT was observed. Other toxicities reported at dose level 2 included grade 1 hypermagnesemia and grade 1 alkaline phosphatase elevation.

At dose level 3, three eligible patients were evaluated for toxicity, and none of them experienced a DLT. One patient reported treatment-related diarrhea (grade 1) and another reported treatment-related nausea (grade 1).

Clinical responses

RECIST (Response Evaluation Criteria in Solid Tumors) criteria were applied for response assessment. In addition to the initial interpretation by a Mayo Clinic radiologist, central radiology review of all computed tomography images and positron emission tomography (PET) images was performed by two of the authors (S.K.C. and V.L., respectively). Eleven of the twelve patients showed progressive disease as the best objective response at the time of their first evaluation (day 28 for dose levels 1 and 2, and day 42 for dose level 3). One patient in the dose level 2 group had stable disease by imaging on day 28; however, he showed symptomatic deterioration with significant decline in his performance score and increase in the tumor marker CA19.9 by 88%. The median time to progression, as measured from entry to the study, was 32 days (range 23–42 days). There was a statistically significant increase in tumor volume: median increase 50.7%, mean increase 204.5%, P= 0.0010. In addition, the tumor marker, CA19.9, also increased significantly: median increase 134.9%, mean increase 462.7%, P= 0.0010. We also examined whether assessment of response by measurement of tumor Hounsfield units, as applied by Choi et al. in gastrointestinal stromal tumors^7,8 could yield a better prediction of outcome as compared to response assessment by using Response Evaluation Criteria in Solid Tumors criteria. There was no correlation between change in Hounsfield units following treatment and time to progression (Pearson correlation coefficient 0.39, P value = 0.23) or survival (Pearson correlation coefficient 0.02, P value = 0.94), thereby indicating that assessment of Rexin-G response in pancreatic cancer patients by measuring tumor Hounsfield units represents an unreliable method of predicting outcome.

In addition, all the patients had fluorodeoxyglucose PET scans performed at baseline and at the time of the first post-treatment evaluation. There was disease progression with increase in size/fluorodeoxyglucose uptake of existing lesions and development of new lesions in 10 of the 12 patients. One of the twelve patients had stable disease as shown by PET, and another developed massive ascites that obscured accurate post-treatment PET assessment of abdominal metastases. The median increase in PET standardized uptake values was 36.3% (range: 11.7–68.7%), P = 0.0244. These data collectively indicate lack of antitumor activity.

Survival

The median survival was 3.5 months from the initiation of the study (range: 1.6–6.9 months). One of the twelve patients, currently on chemotherapy with Taxol, gemcitabine, and capecitabine is still alive at 6 months after initiation of the study.

Viral kinetic studies

Serum samples were collected at baseline, and at 5, 30, 60, and 120 minutes, and 24 hours after vector infusion on day 1 and tested for the presence of Rexin-G vector. The analysis was performed by Epeius Biotechnologies. The neo^r-selectable Rexin-G vector levels were very low (<1 × 10²) but detectable in blood samples obtained 5 minutes after Rexin-G infusion in all three patients at dose level 1, in two of the six patients at dose level 2, and in two of three patients at dose level 3. The vector was still detectable at 30 minutes in two of the three patients at dose level 1, none of the six patients at dose level 2, and in none of the 6 patients at dose level 3 (Table 3). No vector was recovered at any time point beyond 30 minutes.

Table 3.

Viral kinetics after intravenous (IV) administration of rexin-G

		Vector recovery after IV administration
Patient	Dose level	5 minutes	30 minutes
1	1	34 CFU/ml	6 CFU/ml
2	1	50 CFU/ml	9 CFU/ml
3	1	60 CFU/ml	0
4	2	0	0
5	2	0	0
6	2	0	0
7	2	30 CFU/ml	0
8	2	9 CFU/ml	0
9	2	0	0
10	3	8 CFU/ml	0
11	3	0	0
12	3	0	0

Abbreviation: CFU, colony forming units.

Detection of anti-vector antibodies in patients’ sera

Testing for the presence of vector-neutralizing antibodies was performed at 4 weeks (dose levels 1 and 2) and 6 weeks (dose level 3). No Rexin-G neutralizing antibodies were detected in any of the patients.

Testing for the presence of replication-competent retroviruses in patients’ PBMC

Peripheral blood mononuclear cells (PBMCs) in five of the six patients at dose level 2 and two of three patients at dose level 3 were tested for the presence of replication-competent retroviruses (RCR) using real-time polymerase chain reaction (PCR), either at week 4 (dose level 2) or at week 6 (dose level 3). All the samples that were tested were found to be negative for replication-competent retroviruses.

Vector DNA integration studies

Vector integration in PBMCs DNA was tested at weeks 1 and 4 for patients at dose levels 1 and 2, and at day 5 and week 6 for patients at dose level 3. No vector integration in PBMC DNA was observed in any of the patients.

Discussion

Pancreatic cancer is fourth in the list of leading causes of death due to cancer, with an estimated 33,000 deaths from this cause in the U.S. in 2007 (ref. ⁹). Gemcitabine has become the standard of care for the treatment of advanced/recurrent disease since the late 90s, when a randomized phase III trial demonstrated its superiority over 5-fluorouracil¹⁰. Recently, the combination of the tyrosine kinase inhibitor erlotinib with gemcitabine was approved by the Food and Drug Administration for treatment of advanced pancreatic cancer, on the basis of a phase III National Cancer Institute of Canada trial that showed a modest survival benefit for the combination arm.¹ There is no standard second line treatment for this disease that can convincingly extend survival, and the need for developing novel therapeutic approaches is obvious.

Cyclin G1, a transcriptional target of p53 that is induced by DNA damage, plays an important role in regulating the cell cycle and it is overexpressed in different malignancies including pancreatic cancer.⁴ Rexin-G is a pathotropic nonreplicating viral vector that expresses a dominant negative cyclin G1 construct, and is engineered to display a von Willebrand factor–derived collagen-binding motif at its amphotropic 4070A envelope protein. The displayed collagen-binding motif has the potential of directing the vector to areas of active collagen remodeling, including tumor stroma. In preclinical work, Rexin-G has demonstrated antitumor activity against pancreatic cell lines, and decrease in the size of liver metastases in a metastatic pancreatic cancer model after portal vein administration.^3,5

The initial data regarding the use of Rexin-G in the treatment of pancreatic cancer in humans derive from the Philippines. Tumor stabilization after administration of doses ranging from 2.7 × 10¹⁰ to 3 × 10¹¹ CFU with duration ranging from 3.4 to >5.5 months were reported.⁶ In a subsequent overview of the 3-year clinical experience in the Philippines, the results of three clinical trials were summarized; five of the six patients in the first pancreatic study were reported to have shown a partial response, with progression-free survivals ranging from 2 to 9 months and a median survival of 24 months.¹¹ Five of six patients had failed standard chemotherapy. The virus dose employed for five of six patients was 2.5 × 10¹¹ U per cycle, and patients received from one to seven cycles of treatment.¹¹ The sixth patient received 2.28 × 10¹⁰ U, followed by gemcitabine. A second trial in patients with recurrent solid tumor included three patients with pancreatic cancer, one previously untreated. The virus dose in this trial was 6 × 10¹¹ CFU per cycle. Two of these three patients were reported to have achieved partial responses. Progression-free survival was 3 months for one of these patients and 6+ months for the other. Overall survival in the group of three patients with pancreatic cancer was 6+ months, 9 months, and 11+ months.¹¹ In a third trial, a “calculus of parity” approach was followed for estimating the optimal virus dose in three patients, two of them with pancreatic cancer, who received doses up to 2.5 × 10¹² CFU. Antitumor responses were reported in both patients,¹¹ but no further details were provided. On the basis of these data, Rexin-G was approved as an anticancer agent in the Philippines. Since 2003, it has also obtained orphan drug status in the U.S. (http://www.FDA.gov). Those initial clinical data served as the rationale for a confirmatory phase I/II Rexin-G trial in pancreatic cancer patients.

Our trial is the first trial in the United States involving IV systemic administration of a retroviral vector. The treatment was well tolerated. The only DLT was liver function tests elevation in one patient who received 1 × 10¹¹ CFU (dose level 2), and this resolved within 4 days of discontinuing the treatment. No DLT was observed when a newer vector preparation was administered to the three-patient expansion cohort in dose level 2, and to three dose level 3 patients, despite the omission of the premedications including Benadryl and hydrocortisone. The fact that there was no evidence of RCR and retroviral integration in patients’ PBMC further underlines the safety of the approach.

The doses employed in our trial ranged from 1 × 10¹¹ to 6 × 10¹¹ CFU, which is the dose range in which antitumor activity occurred in two of the three published trials from the Philippines. However, no antitumor activity was observed in our trial; 11 of the 12 patients treated in our study showed progressive disease as the best objective response. The median time to progression was 32 days and the median duration of survival from treatment initiation was 3.5 months, both of which are significantly shorter than those reported in the two trials in the Philippines, in which comparable doses of the virus were used. This difference in antitumor efficacy could theoretically be explained on the basis of pharmacogenomic differences between Asian and non-Asian populations of patients, and the heterogeneity of the Philippines study groups, which included untreated patients and patients who concomitantly received gemcitabine. The small sample sizes (11 pancreatic cancer patients in the published Philippines trials and 12 patients in our trial) prohibit definitive conclusions.

These results also emphasize the importance of independent multicenter clinical trials in clearly defined patient populations in order to objectively assess the safety and efficacy of novel antitumor agents. On the basis of our data, further testing of Rexin-G in these dose ranges for the treatment of pancreatic cancer patients cannot be recommended.

MATERIALS AND METHODS

Inclusion/exclusion criteria

Inclusion criteria for patients were (i) the presence of locally advanced or metastatic pancreatic cancer resistant to gemcitabine chemotherapy, as indicated by disease progression ≤6 months from the last gemcitabine treatment; (ii) histological or cytological confirmation at diagnosis or recurrence; (iii) an Eastern Cooperative Oncology Group performance score of 0–1 and adequate hematologic, hepatic, and kidney function.

The exclusion criteria included: (i) human immunodeficiency virus, hepatitis B virus, or hepatitis C virus positivity; (ii) clinically significant ascites, or medical or psychiatric conditions that could compromise successful adherence to the protocol; and (iii) unwillingness to employ effective contraception during treatment with Rexin-G and for 4 weeks following the completion of treatment. The protocol was approved by the Mayo Clinic Institutional Review Board and informed consent was obtained from all the study participants.

Study drug

Rexin-G is a matrix (collagen)-targeted retroviral vector derived from the MoMLV.² The vector encodes an N-terminally deleted mutant of cyclin G1, transcribed from the viral long-terminal repeat and a bacterial Neomycin resistance (neo^r) gene transcribed from the viral simian virus 40 early promoter.¹² The Rexin-G vector is produced by transient co-transfection of human embryonic kidney 293 T cells. Clinical vector production and characterization have been described elsewhere.¹¹ Clinical grade vector (provided by Epeius Biotechnologies) was stored at −70 ± 10 °C, and thawed in a 37 °C water bath 15–30 minutes prior to use. Titers of viral preparations used for the clinical trial ranged from 2 × 10⁷ CFU/ml to 5 × 10⁹ CFU/ml.

Pretreatment evaluation and follow-up studies

Pretreatment evaluation included history, physical examination, hematology group, chemistry group, assessment of coagulation (fibrinogen, soluble fibrin monomer, prothrombin time, and activated partial thromboplastin time), testing for human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, imaging evaluation including computed tomography or magnetic resonance imaging of the chest, abdomen, and pelvis, fluorodeoxyglucose/PET scan, chest X-ray, and CA19.9. Patients in dose levels 1 and 2 had complete blood counts and chemistry groups testing performed on a daily basis before virus administration and on days 1 and 7 and 15–21 after administration, while coagulation parameters were assessed on alternate days. In addition, toxicity was assessed daily before virus administration, and on days 10, 24, and 28 after administration. Efficacy assessment using imaging studies, and measurement of tumor markers was performed on day 28. The PBMCs from patients were collected for assessment of viral integration on days 7 and 28. In addition, viral kinetic studies were performed at baseline, 5 minutes, 30 minutes, 1 hour, 2 hours, and 24 hours after completion of the first virus infusion. Real-time PCR to detect the presence of RCR in PBMCs was performed on day 28. Patients at dose level 3 had baseline assessments similar to those at dose levels 1 and 2. Toxicity assessment, complete blood counts, and chemistry group were performed on days 1, 5, 8, 15, 22, and at week 6. Coagulation parameters were assessed at baseline, on days 8, 15, and 22, and at week 6. Viral kinetics were assessed on a similar schedule as for dose levels 1 and 2. Viral integration in PBMCs was assessed on day 5 and week 6 and PCR for the presence of RCR was performed in PBMCs at week 6. Assessment of efficacy, with computed tomography or magnetic resonance imaging of the chest, abdomen, and pelvis, PET scan, and determination of CA19.9 levels, was performed at week 6.

Treatment schedule

Three dose levels of the virus were tested—dose level 1: 7.5 × 10⁹ CFU per day on days 1–7 and 15–21, amounting to a total dose of 1 × 10¹¹ CFU per cycle; dose level 2: 1.1 × 10¹⁰ CFU per day, days 1–7 and 15–21, for a total dose of 1.5 × 10¹¹ CFU per cycle; dose level 3: 3 × 10¹⁰ CFU days 1–5, 8–12, 15–19, 22–26, amounting to a total dose of 6 × 10¹¹ CFU per cycle. For patients at dose level 3, a second cycle could be administered if, at the time of evaluation at week 6, there was no evidence of progressive disease and toxicity was acceptable. An additional dose level consisting of 8 × 10¹⁰ CFU on days 1–5, 8–12, 15–19, and 22–26 for a total dose of 1.6 × 10¹² CFU per cycle was initially planned, but it was never activated because of insufficient availability of the drug. Patients at dose level 1 and the first group of three patients at dose level 2 received the following premedications prior to the viral treatment: Benadryl (12–50 mg) IV, Tylenol 500–1,000 mg po, hydrocortisone 15–100 mg IV, and Demerol 15–50 mg IV. The last three patients at dose level 2 and all the patients at dose level 3 received a higher-purity preparation of the Rexin-G vector, and hydrocortisone and Demerol were omitted before treatment with the virus.

Statistical methodology

The study employed the standard “cohort of three” design.¹³ Three patients were treated at each dose level with expansion to six patients per cohort if DLT was observed in one of the three initially-enrolled patients at each dose level. The maximum tolerated dose was defined to the highest safely tolerated dose, where not more than one patient experienced DLT with the next higher dose level having at least two patients who experienced DLT.

DLT was defined as any grade 3–5 adverse event considered possibly, probably, or definitely related to Rexin-G, excluding grade 3 absolute neutrophil counts lasting <72 hours, grade 3 alopecia, or any grade 3 or worse nausea, vomiting, or diarrhea where the patient did not receive maximal supportive care (National Cancer Institute Common Terminology Criteria Version 3.0).

Frequency tables, graphs, and summary statistics were used for describing toxicity and outcome data. In addition, Kaplan–Meier methodology¹⁴ was used for describing the distribution of time-to-disease progression, and survival.

Toxicity/response criteria

Toxicity was graded using the National Cancer Institute Common Terminology Criteria Version 3.0. Response was evaluated by computed tomography and/or magnetic resonance imaging of the chest, abdomen and pelvis, performed at baseline and after each treatment cycle. Tumor response was evaluated using RECIST criteria.¹⁵ Fluorodeoxyglucose/PET scan was also obtained in all patients at baseline and after each treatment cycle, as were levels of the tumor marker CA19.9, and these were employed as additional parameters in response assessment.

Correlative laboratory analysis

Correlative laboratory analysis was performed in the Epeius Biotechnologies Quality Control Unit.

Viral kinetics studies

Rexin-G vector levels¹⁶ were measured from serum samples obtained at 0, 5, 30, 60, and 120 minutes and 24 hours after vector infusion on day 1. Rexin-G vector concentration (viral titer) was determined and quantified on the basis of expression of the neomycin resistance (neo^r) gene product. Briefly, 1.5 × 10⁴ HT1080 (human fibrosarcoma) cells were plated in each of 12-well plates one day before transduction. The culture medium was incubated with 0.5 ml of serial dilutions of viral super-natant with 8 μg/ml polybrene for 3.5 hours at 32 °C, 5% CO₂, with gentle rocking. 0.5 ml of fresh media was added to the cultures, which were then maintained overnight at 37 °C, 5% CO₂. For expression of the neo^r gene product, G418-resistant colonies were selected by treatment with G418 drug (500 μg/ml) beginning 24 hours after transduction. The G418-resistant colonies stained with methylene blue were quantified by limiting dilution after incubation in G418 drug for 13 days. Viral titer was expressed as the number of CFU per milliliter of serum (CFU/ml).

Detection of anti-vector antibodies in the sera of patients

Testing for the presence of anti-vector antibodies was performed on serum samples obtained preinfusion and at 4 weeks (dose levels 1 and 2) or 6 weeks (dose level 3) after initiation of the treatment. The presence of anti-vector antibodies was tested for, using vector neutralization assays combined with Western slot blot analysis.

Vector neutralization assay

A375 cells, a human amelanotic melanoma cancer cell line (American Type Culture Collection), were incubated in 12-well plates, at a plating density of 0.8 × 10⁴ cells per well, in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (D10). After overnight attachment, the cells were exposed to 1 ml of a mixture of Rexin-G vector and the patient’s pre- and post-treatment serum, fetal bovine serum and D10 (as controls) in the presence of polybrene (8 μg/ml) for 2 hours at 37 °C, 5% CO₂. One milliliter of fresh D10 was added, and the cultures were further incubated at 37 °C, 5% CO₂. The medium was replaced with 1 ml of fresh D10 the next day. In order to assess the dominant negative cyclin G gene product potency of the Rexin-G vector, the transduced cells were evaluated for their reduced proliferative potential by counting the number of viable cells in triplicate cultures at serial intervals (4 and 5 days) after transduction, and applying the calculation: % vector inhibitory activity (vector + D10):

$$\frac{\text{No.}\text{of}\text{cells}(\text{D}10)-\text{No.}\text{of}\text{cells}(\text{vector}+\text{D}10)}{\text{No.}\text{of}\text{cells}(\text{D}10)}\times 100$$

In order to obtain the vector inhibitory activity in serum-treated and control cultures, the mean cell numbers in cultures transduced with mixtures of vector + D10 were compared with vector + serum-treated cultures and expressed as % vector inhibitory activity, using the formula: serum % vector inhibitory activity (vector + serum):

$$\frac{\text{No.}\text{of}\text{cells}(\text{D}10)-\text{No.}\text{of}\text{cells}(\text{vector}+\text{serum})}{\text{No.}\text{of}\text{cells}(\text{D}10)}\times 100$$

The presence of vector-neutralizing activity is detected by comparing the % inhibitory activity in mixtures of vector + D10 versus vector + serum, using the formula: % Vector neutralizing activity: % inhibitory activity (vector + serum) − % inhibitory activity (patient baseline or fetal bovine serum). A negative value indicates a drop in inhibitory activity. A value ≤10 indicates that no vector neutralization has occurred, and a value >10 indicates that neutralization has occurred.

Testing for antibodies against the gp70 env protein

The detection of serum antibodies against gp70 was conducted by Western slot blot analysis using gp70 containing Rexin-G retroviral vector as a polyacrylamide gel electrophoresis resolved capture antigen incubated with either sera from treated patients or positive control gp70 murine monoclonal antibody.¹⁷ The appearance of an immunoreactive band on the Rexin-G slot blot at 70 kd (gp70) in slot lanes of a patient’s postinfusion serum indicates the presence of postinfusion antibodies against the gp70 env protein.

Testing for the presence of RCR in patient peripheral blood lymphocytes

Testing for the presence of RCR was performed on DNA extracted from peripheral blood lymphocytes of patients, obtained at baseline (before vector infusion) and at either 4 weeks (dose levels 1 and 2) or 6 weeks (dose level 3) after treatment. The assay was designed to use PCR to detect the presence of RCR DNA sequences after transfusion. In this study, real-time PCR with an iCycler, Master mix reagents and optimized MoMLV Env primers is used for amplifying a small portion of the 2,001-bp MoMLV envelope (MoMLV Env) gene (164-bp fragment from 411–574 bp) present in the Rexin-G retroviral vector. Serial tenfold dilutions of control MoMLV Env plasmid BVE-RVE DNA in the picogram to femptogram range were diluted into control samples of human genomic DNA to establish a standard curve down to the limit of detection of the assay. SYBR green DNA intercalation dye was used as a probe to monitor the progress of increased DNA quantities generated in the PCR. MyIQ software was used for recording the real-time PCR data and calculating the standard curve as well as the values for the samples taken from patients. The real-time PCR results for amplification of the MoMLV Env gene from 3 μg DNA had to be below the lowest result on the standard curve and/or below preinfusion values to be considered negative for RCR.

Vector DNA integration studies

Testing for the presence of vector DNA integration was performed on DNA extracted from peripheral blood lymphocytes obtained preinfusion, and at either 1 week and 4 weeks (dose levels 1 and 2) or at day 5 and 6 weeks (dose level 3) after initiation of the treatment. Testing for vector DNA integration in peripheral blood lymphocytes was performed by centrifuging each patient’s blood sample in a CPT collection tube to separate white blood cells from red blood cells and serum. White blood cell DNA was isolated using a Qiagen blood DNA isolation kit, then quantified using a ultraviolet spectrophotometer. Real-time PCR with an iCycler, Master mix reagents and optimized neo primers were used for amplifying a small portion of the 795-bp neomycin phosphotransferase gene (75-bp fragment from 382–456 bp) present in the dnG1-Erex retroviral vector. Serial tenfold dilutions of Rexin-G retroviral vector control plasmid dnG1-Erex DNA in the picogram to femptogram range were diluted into control human genomic DNA in order to establish a standard curve down to the limit of detection of the assay. SYBR green DNA intercalation dye was used as a probe to monitor the progress of increased DNA quantities generated in the PCR. MyIQ software was used to record the real-time PCR data and calculate the standard curve as well as the value for samples taken from the patients. The real-time PCR results for amplification of the neomycin phosphotransferase gene from patient lymphocyte genomic DNA had to be below the lowest result on the standard curve and/or below preinfusion values to be considered negative for vector integration.