Supplementary Methods
Ad35K++ manufacturing: The Ad35K++ construct for expression in E.coli has been described previously.9
Ad35K++ was produced using a 4L fermentor under GLP condition by the Fred Hutchinson Cancer
Research Center Biologics Production Core Laboratory. A single vial from the FHCRC research bank of
pQE30-Ad35K++ is used for the fermentation production.
Fermentation: A scrapping from the vials is performed and placed in warmed 25 ml LB Broth with 100
ug/ml ampicillin in a 125 ml Erlenmeyer flask (starter culture). The flask was placed in the shaker
incubator with the settings of 250 rpm, at 37.1 C overnight. The 4L fermentor was prepared, pH probe
calibrated and the system sterilized by autoclaving with water. Following autoclaving, the vessel was
maintained under positive pressure. On the following day, 400 ml of LB broth with 100 µg/ml ampicillin
was added to a 2L Erlenmeyer flask (seed culture). Media was warmed for approximately 20 to 30
minutes. The overnight culture was checked for growth using spectrophotometry and the amount added
to the seed culture adjusted to an optical density at 600 nm of 0.05. The fermentor was aseptically filled
with 4 L of LB Broth (Invitrogen) and 50% autoclaved antifoam, 20% sterile filtered phosphoric acid and
sterile 5 N NaOH connected for level, acid and base control. Ampicillin was then added to a final
concentration of 100 µg/ml alongside 80 mL of 50% sterile glycerol. Finally, parameters were set to
control to 10% air (2% oxygen), pH 6.8 and 37.1° C. The impellor was set with range of 300 to 800 rpm
and was used to maintain dO2 levels with increased agitation. At 800 rpm, if the stirrer was unable to
maintain dO2 settings of 10%, O2 was used for supplementation. Once seed culture reached an OD 600
of 0.5 to 1.0, the 7 L fermentor was seeded at 4 L working volume to a calculated optical density at 600
nm of 0.05. On day 3, the fermentor was checked for growth and inoculated with 1mM Isopropyl β-D-1thiogalactopyranoside (IPTG) to induce protein expression. After induction, fermentation was continued
for 6 hours, and then bacterial cells harvested by centrifugation using an SLA 3000 rotor at 8000 rcf for
15 minutes. Containers were weighed to assess net pellet wet weight. Lysis buffer was added to pellets
at 1 to 2 ml per gram of pellet. Bacterial cells were suspended and stored at -70 °C.
Nickel affinity purification: 20 mL of immobilized metal affinity chelate (IMAC) matrix was rinsed with
water and charged on column with a 200 mM solution of nickel chloride in phosphate buffer. The matrix
was then washed, rinsed and equilibrated with Lysis buffer consisting of 50mM NaH2PO4, 300 mM NaCl,
and 10mM imidazole, pH 8.0. Matrix was removed and suspended in 50% slurry in Lysis buffer. Bacterial
pellets were removed from storage and thawed in a room temperature water bath. Lysozyme was
added to a final concentration of 1 mg/ml and incubated on ice for approximately 30 minutes. The
proteinase inhibitor PMSF was added to a final concentration of 1 mM. Cell pellets were then placed in a
blender to mix and break up any remaining clumps, and then microfluidized at 15,000 psi. The
microfluidized bacterial debris was clarified by centrifugation at 8000 rcf for 20 minutes. Supernatant
was collected and checked for pH and adjusted appropriately. The supernatant was then filtered using a
0.45 µm filter. The Ni-charged matrix was added to filtered supernatant and incubated overnight at 4 °C
while on an orbital shaker. The supernatant and matrix were collected and spun in 500 ml tubes to
pellet the matrix. The matrix was suspended and placed into 10 mL columns and the supernatant
flushed through the matrix at a rate of 3ml/min or a linear flow rate of 36.7 cm/hr (residence time 6.5
min). When complete, the matrix was washed with approximately 5 column volumes of Lysis buffer and
then with 50 column volumes of Wash buffer (50mM NaH2PO4, 300 mM NaCl, 60 mM imidazole, 20%
glycerol pH 8.0). Ad35K++ His Tagged protein was eluted with Elution buffer (50mM NaH2PO4, 300 mM
NaCl, 250 0mM imidazole, 20% glycerol pH 8.0), and collected in single column volume fractions. Each
fraction was checked for absorbance at 280 nm and using extinction coefficient of 2.29 to determine
concentration and total protein. Pooled fractions were dialyzed overnight against three exchanges of
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PBS) using 10 kDA molecular weight cut-off dialysis membrane. Dialyzed product was collected and
sterile filtered through a 0.2 µm filter.
Removal of endotoxin: A QFF Sepharose column (5 cm × 7 cm) was packed and sanitized for purification
of his tagged AD35K++ protein utilizing 1N NaOH, water for injection (WFI) and 2M NaCl. The QFF
column was then equilibrated with half strength PBS. Samples were submitted for determination of
system and column endotoxin levels. Endotoxin levels were found to be less than 0.5 EU/ml. Ad35K++
was then diluted 1:1 with WFI, and then loaded onto the QFF column. No flow through product was
detected. Column was then rinsed with 5 column volume of half strength PBS. Ad35K++ was then eluted
from the column with full strength PBS. Ad35K++ HT was then transferred to an Amicon Pressure cell
with a 10 kDA membrane for buffer exchange and concentrated to 2 mg/ml. The final product was
filtered through a 0.2 µm filter. Ad35K++ was aliquoted at 0.5 ml per tube (1.8 ml Nalgene cryotube)
using a Flexicon PF6 programmable peristaltic pump with a dispensing tubing set. Once completed,
AD35K++ product was stored at -80 °C.
Generation of C38C13-hCD46/CD20 cells. To generate C38C13-hCD46/CD20 cells, we produced VSVGpseudotyped lentivirus vectors containing the cDNAs for human CD46 or CD20 under the control of the
EF1 promoter and transduced the mouse lymphoma cell line. A total of 100 colonies stably expressing
the transgenes were screened by flow cytometry for surface human CD20 and CD46 levels and one
clone with levels that were comparable to human lymphoma (Raji) cells was selected for in vivo studies.
ELISPOT assay: hCD46/CD20 transgenic mice were intravenously injected with PBS and Ad35K++
(2mg/kg). Three days later, mice were intramuscularly vaccinated with 5×109 pfu of Ad.HBeAg
expressing the HBVe antigen.14 Twelve days later, spleen cells of naïve syngeneic animals were obtained
and pulsed with 5μg/ml of recombinant HBe or HBsAg antigen. On day 14, vaccinated animals were
sacrificed, their spleen cells were collected and 1×106 cells were mixed with 1×106 of ex vivo pulsed
splenocytes for in vitro sensitization (I.V.S.). After 24 hours of incubation in 96 well plates, cells were
plated at a concentration of 105 cells/100 μl into wells of 96-well hydrophobic polyvinylidene difluoridebacked plates (Millipore, Bedford, Mass.), previously coated with 50μl of 10μg/ml anti-IFNγ monoclonal
antibody (1-D1K, mouse immunoglobulin G1; Mabtech, Nacka, Sweden) overnight at 4°C. As a positive
control, phytohemagglutinin (Sigma, St. Louis, MO) was added to two wells at a final concentration of 1
μg/ml. All responses were tested in triplicate. Plates were incubated overnight, washed with PBS
containing 0.05% Tween 20, and incubated at room temperature for 2 hours with biotinylated anti-IFNγ
monoclonal antibody at 1μg/ml (7-B6-1, mouse immunoglobulin G1; Mabtech). Binding was developed
using the Vectastain ABC Elite kit (PK-6100; Vector Laboratories, Burlingame, CA).
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